Seas pretw pe Fa rGeere OTST SREB RENAE as So eeress : 7 on {Boo yan cd pease re ~ < <= : sti z : eres tagegetetsh aS ‘ + Pet it eed 7 ene SoS eae sateen ae SoS Seaesaee SSSETS Seeaeeet Sa ree ce StS SSyreste etnias ere ne te eae eaties : == : : ‘ - - : 3 restless Jaen : 2 wtdndaa a {ih See mane sa t a peecritae agbeeteneee Mt) rhivdat iyt PrTebey taphiet tere hoe) : 4 sine nae sit fai tee Brit reed Cpagbneds 1th itert Sina) eiystotiteert aeeverebene unit gerracee it fue PEPDELP AVG aie ate Heigeeites t Tienes ttt TOM tty ent vhtine bith Hae teeseireritie rodhielea feetrati esi tt H ; Yeyitrs ftir eengredy setirtiy heal ites eheghenue pieyvbecvipess MADPLEAL ELA! ( yu { settee irtet trys Hy MUL it itpetee rgatr in they ehtagt 4 iti yepieire Pieungboapatasgebre feedirsretetarr hurt faves shea tt} af treed POheeae NyTITITG yeterbiaa wheeatavatea siyrteenty tit teeieretepeeeet tgagertactacdrieny tthe s2) e crrtaqyeerreeretenieriees Vanier t yaeetty ; puede i ares dares t a) bad Hite t 1 patheit esate qierateret tet 3? ree behbasael Peepereeiyh TEPD Re eT pata yet yet UREEPLS ECR) corp a bse e rel intone rh Tey reneteqiye t il if] Hn ttogerertraretoade pete a 1 he Ww susrhey peyare ieqierge ttetaet rhitis Trerttes tarervenieey tt { tetrgeier Cherdtredgere eiereaedd Se ee Quintin oto em Sn ens a ng nr ors ¢ % Er ye re > yrpdgreert eas f PR IKI s oh a ad a! at a Si a saan ee eee Scales % GEC hn = res Poe eel fin feene eo he heh hy tPF es i tho 200" . Omg y pe se. ‘yy, O4e ices THE AMERICAN JOURNAL OF ANATOMY EDITORIAL BOARD CHARLES R. BARDEEN G. Cart HUBER J. Puayrain McMurricu University of Wisconsin University of Michigan University of Toronto Henry H. DonaLpson GeorGE 8S. HUNTINGTON CHARLES 8. Minot? The Wistar Institute Columbia University Harvard University Simon H. GaGe FRANKLIN P. Mau Georce A. PIERSOL Cornell University Johns Hopkins University University of Pennsylvania Henry McK. KNower, SECRETARY University of Cincinnati VOLUME 14 1912-1913 THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA. CONTENTS 1912-19138 -No. 1. NOVEMBER, 1912 H. Hays Butuarp. On the interstitial granules and fat droplets of striated CPE ay UAE ESET UTI Sas Rea pee > SRS Oe oot Oe 1 Apmont H. Crarx. On the fate of the jugular lymph sacs and the develop- ment of the lymph channels in the neck of the pig. Four figures....... 47 R. H. Waiteneap. On the chemical nature of certain granules in the inter- stitial cells of the testis. Six figures.. x . 683 PrarL Brices BuLLarD. A comparative ete of fhe mee nent regions of the spinal cord in a series of mammals. Twenty-five figures......... 73 E. Linpon Metuus. The development of the cerebral cortex. Two figures 107 No. 2. JANUARY, 1913 CHARLES EUGENE JoHNSON. The development of the prootic head somites and eye muscles in Chelydra serpentina. Twenty-four figures (ten plates).. 119 FRANKLIN PARADISE JOHNSON. The development of the mucous membrane of the large intestine and vermiform process in the human embryo. SIR UIE IEC Cee wo co Tu We ARE eae Sone eR gee eee feed 187 FRANKLIN PARADISE JOHNSON. The effects of distention of the intestine upon shape of villi and glands. Rleven figures... <. os ...% ~~ <0.<5~0% + + S208 235 E. Victor Smitx. Histology of the sensory ganglia of birds. Forty figures. 251 No. 3. MARCH Atwin M. PAppENHEIMER. Further studies of the histology of the thymus. Five plates......... te eee eee eee eee 299 Ricwarp E. ScaMMon. The development of the elasmobranch liver. I. The early development of the liver. II. The development of the liver ducts and gall bladder. Fifty-four figures..................--.---.-5--- 333 ill iv CONTENTS No. 4. MAY S. Waiter Ranson. The fasciculus cerebro-spinalis in the albino rat. Ten QUT ES AC i wire vag htolece a's bes MAR OO Oils Asia ge ee 411 C. W. Prentiss. On thedevelopment of the membrana tectoria with reference to its structure and attachments. Fourteen figures..................... 425 H. L. Wreman. Chromosomes in man. Ten figures........................ 461 JEAN ReDMAN Otiver. The spermiogenesis of the Pribilof fur seal (Cal- lorhinus alascanus J. and C). Thirty-eight figures..................... 473 ON THE INTERSTITIAL GRANULES AND FAT DROP- LETS OF STRIATED MUSCLE H. HAYS BULLARD From the Anatomical Laboratory, Tulane University of Louisiana SEVEN FIGURES CONTENTS ImeViatenialeamcdimethodst-mccntessesoke era ce coi tn acaehoe aEbnid Gave us Pate eae 3 lees omrenclatumerera crs oe at eee ween eer Ty kee Uh, Wh Se eee 5 III. Relation of interstitial granules and fat droplets to color and structure COLETINUIS CG Pear ke eth ay t ee rat NE a nh an RM es sae 6 a. Light and dark muscle fibers b. Relation of light and dark muscle fibers to white and red muscle ce. Morphology and position of fat droplets . d. Morphology and position of true interstitial granules IV. General occurrence of interstitial granules and fat droplets............ 18 VY. Chemical nature of interstitial granules and fat droplets.............. 21 a. Chemical nature of true interstitial granules 1. Refractive character 2. Solubility 3. Results with Cresylviolett R R, and Cresylechtviolett 4. Results with the methods of Weigert, Altmann, Benda and Regaud Results with acid fuchsin . Results with Sudan 111, osmium tetroxide, and gold chloride 7. Summary b. Chemical nature of fat droplets 1. Refractive character: Double refraction . Solubility Results with Scharlach R and Sudan 111 Results with osmium tetroxide Results with the Nile blue method . Results with the methods of Benda, Fischler, and Klotz for free fatty acids and soaps 7. Results with the Weigert method and related methods 8. Formalin fixation 9. Summary o> Or aoe wh VI. Physiological significance of interstitial granules and fat droplets.... 39 Pie SMM ATy. anid CONCLUSIONS): ., > erRici.. sie cays wersaces Sets sek sae eae a 42 inihetsay Ue merue Cit eeen |. Meare S/o nee eee Re ree. oe tot, Cit cots es A4 1 THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, No. 1 NOVEMBER, 1912 bo H. HAYS BULLARD The granules to be found between the myo-fibrils or muscle columns of cross striated muscle, although mentioned by Henle (41) were first described in detail by K6lliker (57) who called them ‘interstitial granules.’ He applied the term to both fat droplets and true interstitial granules, the latter being of a non- fatty nature. This paper presents observations concerning the structure of striated muscle with especial reference to interstitial granules and fat droplets, including also a brief discussion of their general occurrence, chemical nature, and physiological significance. A number of important communications dealing with the interstitial granules have included a somewhat compre- hensive review of the literature, namely, Retzius (90), Arnold (09), Holmgren (’10), Prenant (’11), and Bell (11). As several of these papers are of recent date I have thought best to omit a chronological review and shall discuss the literature only in so far as its subject matter has a direct bearing on the topics treated in this paper. According to K6élliker both fat droplets and true interstitial granules are of wide distribution, occurring in vertebrate muscle and also in insect muscle. A few observers have denied the exist- ence of two general types of interstitial granules, especially in vertebrate muscle, but usually the work of Koélliker has been con- firmed in this respect. My observations are in accord with those of Kolliker and I shall likewise designate the types of granules as true interstitial granules and fat granules or fat droplets. For the present it may be said that true interstitial granules, at least for the most part, are not completely soluble in absolute aleohol and not readily stained by fat stains such as Scharlach R, while the fat droplets are easily soluble in absolute alcohol and take the fat stains. This does not necessarily mean that the true interstitial granules contain no fatty substance nor that the fat droplets are composed wholly of fatty substances. Under normal physiological conditions the muscle fibers of both skeletal muscle and cardiac muscle of vertebrates may, and usually do, contain true interstitial granules as well as fat droplets. GRANULES AND FAT OF STRIATED MUSCLE 3 I. MATERIAL AND METHODS For the most part the material was obtained from the common laboratory animals: frog, mouse, rat, rabbit, cat, dog, pigeon, and from the bat. In a few cases the nutritive condition of the animal was altered by special feeding in the laboratory. Human material was used to a considerable extent and insect muscle was also examined. The methods used in this study have a direct bearing on the chemistry of the granules and fat droplets, and will be discussed in some detail when considering that subject. The methods may be briefly outlined as follows: 1. Examination of fresh material. a. Preparations made without the addition of fluids. b. Preparations mounted in normal saline solution or 1 to 5 per cent solution of potassium hydroxide. 2. Tests of solubility of interstitial granules and fat droplets with alcohol, xylol, and with ether. 3. Examination of preparations stained by various methods. a. Simple alcoholic solutions of Scharlach R, and Sudan m1. b. Herxheimer’s Scharlach R. c. Nile blue sulphate and Nile blue chlorhydrate. d. Cresylviolett and Cresylechtviolett. e. The methods of Weigert, Altman, Benda, and Regaud. Herxheimer’s (01) stain is prepared by dissolving 2 grams of Na OH in 100 ce. of 70 per cent alcohol, Scharlach R then being added to saturation. The solution is filtered into a tightly clos- ing vessel immediately before being used. Frozen sections or teased preparations of fresh tissue, or material used after two to twelve hours fixation in 20 per cent’ formalin, are washed in 60 per cent alcohol, transferred to the stain for three to fifteen minutes, washed in 60 to 70 per cent alcohol twenty to thirty seconds, followed by water, and mounted in levulose or glycerine. If alcohol washing is omitted precipitates are formed. Fat drop- lets are stained red, true interstitial granules and the protoplasm of muscle fibers are not colored. Alum-hematoxylin or Cresyl- echtviolett may be used as a counter-stain for the nuclei. 4 H. HAYS BULLARD Nile blue: Teased preparations or frozen sections of fresh tissue or material used after a fixation of two to twelve hours in 20 per cent formalin, are stained fifteen minutes to two hours in a satu- rated aqueous solution of Nile blue chlorhydrate, washed in dis- tilled water five minutes or more, and transferred to tap water. After five minutes in tap water the preparation should assume a reddish hue. If this does not occur a slight amount of alkali may be added to the water. The preparations are mounted in either levulose, potassium acetate, or glycerine. When Nile blue sulphate.is used it is necessary to add a somewhat greater amount of alkali, to the tap water. Fat droplets are stained red, purple or blue, true interstitial granules are stained blue. Cresylviolett: Fresh material, or material after two to twelve hours fixation in. formalin, is stained in a dilute aqueous solution of Cresylviolett or Cresylechtviolett for ten to twenty minutes, then washed three to five minutes in distilled water and mounted in levulose syrup. Fat droplets are colorless, or (rarely) a faint red or blue, true interstitial granules are blue. For the details of the complicated methodsof Weigert, Altmann, Benda and Regaud, the reader is referred to Encyklopadie der Mikroskopischen Technik, Berlin, Wien, 1903, and to the discus- sion of Fauré-Fremiet, Mayer and Schaeffer (’10). Benda’s method in particular is unnecessarily complex, requiring about two weeks to prepare a specimen, and its results are not uniform. Satisfactory preparations were obtained by a modified Weigert method which may be briefly stated as follows: Fix twenty-four hours or more in a 10 to 20 per cent solution of formalin. (4 to 8 per cent formaldehyde) with the addition of 0.75 per cent sodium chloride, then mordant in 5 per cent aqueous potassium bichromate four to seven days. Imbed in paraffin and section. Stain warm two to six hours in a mixture contain- ing hematoxylin 1 gram and 2 per cent acetic acid 200 ce. Decol- orize by use of Weigert’s differentiating fluid, or by dilute (0.12 per cent) potassium permanganate followed by the oxalic acid- potassium sulphite mixture of the Pal-Weigert technique. Dehy- drate, clear, and mount in balsam. Or prepare paraffin sections, as above, stain in Altmann’s acid fuchsin, decolorize in picric GRANULES AND FAT OF STRIATED MUSCLE 9) acid as used in Altmann’s method and then clear and mount in balsam. The true interstitial granules are stained blue by hema- toxylin or red if acid fuchsin is used. II. NOMENCLATURE In considering the significance of interstitial granules and fat droplets, a clear understanding of the terminology used by various authors is of importance. The true interstitial granules of Kolliker correspond to Altmann’s granules or ‘bioblasts,’ to the ‘mitochondria’ of Benda, and to the ‘plasmasomes’ of Arnold. Granules which do not correspond to the interstitial granules but are concerned in the formation of the myo-fibrills, are also in- cluded by Altmann, Benda and Arnold, as bioblasts, mitochondria -and plasmasomes. A part of the plasmasomes of Arnold, those which are not colored by basic dyes, may represent fat droplets. Benda described mitochondria in fully developed smooth muscle, but thought that they do not occur in the sarcoplasm of adult skeletal muscle, while Holmgren and others, using the technique of Benda, have described such granules in skeletal muscle fibers. The ‘exoplasmic granules’ (J granules and Q granules) and the ‘endoplasmic granules’ of Holmgren correspond to the ‘Sarco- somes’ of Retzius which in turn correspond to Kolliker’s true interstitial granules. It is possible that Retzius and Holmgren may have occasionally confused fat droplets with sarcosomes. Albrecht (’02) classed the interstitial granules with his ‘lipo- somes,’ which could be demonstrated in all tissues by treating fresh preparations with 5 per cent potassium hydroxide. Since some of Albrecht’s liposomes in striated muscle stained by acid fuchsin while others blackened with osmie acid, it is clear that he included as liposomes both the true interstitial granules and fat droplets. Albrecht must have been mistaken in thinking that all his liposomes are seen when fresh tissues are cleared in 5 per cent potassium hydroxide. Fat droplets are brought out clearly but the granules which stain with acid fuchsin are not apparent in such preparations although they may be seen when normal saline is used instead of the alkaline solution. 6 H. HAYS BULLARD Bell (’10, ’11) adopted Albrecht’s term ‘liposome.’ As desig- nated by him, none of the liposomes stain with acid fuchsin but all of them may be colored with Herxheimer’s Scharlach R and all are soluble in alcohol. He describes certain of his liposomes as faintly-refractive and is evidently of the opinion that they corre- spond to the faintly-refractive interstitial granules of Knoll, KOolliker and other observers. I find that the faintly-refractive granules described by Knoll (’80, 791) in the heart and skeletal muscles of the pigeon are readily stained by the acid fuchsin method but do not stain by Herxheimer’s method. These eranules would thus come within the category of liposomes as the term is used by Albrecht, but they form no part of the liposomes of Bell. The faintly-refract- ive liposomes of the latter author are faintly-refractive fat drop- lets and do not correspond to the faintly-refractive granules of Knoll. Knoll himself pointed out that his faintly-refractive granules correspond to the true interstitial granules of Kolliker. The term true interstitial granules will be used in this paper to correspond to the true interstitial granules of Kolliker which as previously mentioned include a part of the bodies described by Altmann, Benda, and Arnold respectively, as bioblasts, mito- chondria and plasmasomes. The term ‘fat’ is here used to include lipoids and cholesterin compounds as well as the fatty acids and their glycerin esters. III. RELATION OF INTERSTITIAL GRANULES AND FAT DROPLETS TO COLOR AND STRUCTURE OF MUSCLE a. Light and dark muscle fibers Krause (64) found that the fibers of the red muscles of the rabbit contain more interstitial granules than are present in the fibers of white or pale muscles. Griitzner (’84) described two types of fibers in human muscles, cloudy or dark and pale or white. He found that all human muscles contain both types of fibers and believed that dark fibers give macroscopically the red appearance to muscles while light fibers correspond to white or pale muscles. , =I GRANULES AND FAT OF STRIATED MUSCLE Knoll (89) concluded that the dark appearance of muscle fibers is due to the presence of interstitial granules, the dark fibers, he states, have many granules while light fibers are free from granules or contain only a small number. Knoll (’91) deseribed the dark fibers as rich in interfibrillar substance or sarcoplasm (‘proto- plasmareich’) and as containing many interstitial granules, while the light fibers were poor in interfibrillar substance (protoplas- maarm’) and contained few granules. As a rule, red muscles contained more dark fibers while white muscles consisted of light fibers to a large extent. The dark fibers were usually of a smaller diameter than light fibers. Knoll also held that in general the active muscles contain a larger proportion of dark muscle fibers with a corresponding increase in the number and size of the interstitial granules. Schaeffer (93) confirmed the work of Griitz- ner and Knoll. He also found that dark fibers showing fixed contraction nodes simulate light fibers. Bell (11) found that, in general, dark fibers contained coarse, strongly-refractive ‘lipo- somes,’ while light fibers contained small faintly-refractive lipo- somes. Hestained the liposomes (fat droplets) with Herxheimer’s solution of Scharlach R and in this way demonstrated the types of fibers. The reader is referred to the papers of Knoll, Schaeffer, and Bell for an account of the general occurrence and distribution of light and dark fibers in different animals. The work of these authors has shown that differences in opacity, corrresponding to hght, dark, and intermediate fibers, occur in the striated muscles of many animals, including practically all those most commonly employed in the laboratory. In my studies the light and dark fibers were easily observed in transverse sections of fresh tissue cut on the freezing microtome. The difference in opacity is most marked when the specimen. is mounted in normal saline and examined with low magnification, the reflected light being cut off by a paper shield or by some other means. The types of fibers were also demonstrated by staining frozen. sections with Herxheimer’s Scharlach R, with Nile blue, or with Cresylviolett. In the pectoral muscles of the pigeon Ss H. HAYS BULLARD where the true interstitial granules are large and numerous, the types of fibers were distinguished by the Weigert and Altmann methods as well as by the other methods just mentioned. The light, dark and intermediate types of muscle fibers are clearly marked in unstained frozen sections of fresh material from normal eats, dogs and rats. If such sections be placed in absolute alcohol for a few minutes and subsequently examined under the microscope the dark and intermediate fibers assume the appearance of light fibers. The fat droplets and also the alco- hol-soluble portion of the true interstitial granules have been removed by the alcohol, and the removal of these droplets and _ eranules causes the dark fibers to lose their opacity. In the dog, eat and rat the true intersitial granules are small and the opacity of the dark fibers is largely due to fat droplets. In the pectoral muscles of the pigeon and the bat, not only fat droplets but also true interstitial granules are an important factor in causing the dark appearance of fibers. Sections of formalin fixed material from the pectoral muscles of the pigeon after treatment with absolute aleohol ten to twelve hours still show the dark fibers much more opaque than the light, while in similar preparations from the dog, cat or rat, dark fibers have lost their opacity and have the appearance of light fibers. The fat droplets have been removed by the alcohol but the large true interstitial granules of the pectoral muscles of the pigeon remain and cause the opaque appearance of the dark fibers to be retained. ‘The staining reac- tions and solubility of fat droplets and true interstitial granules will be dealt with later, but I may here mention that formalin coagulates the true interstitial granules in such a manner as to partially protect them from the action of fat solvents while fat droplets are readily soluble both before and after formalin fixa- tion. Figure 1 represents the types of fibers in the pectoralis major of an exceptionally well nourished white rat. The preparation was stained by Herxheimer’s method. Figure 2 shows a similar specimen from a very emaciated rat. The dark fibers so apparent in the well nourished animal have largely disappeared in the poorly nourished animal. The granules shown in the figures are GRANULES AND FAT OF STRIATED MUSCLE 9 ~« @* 6 ° . ‘ Bra eet 9% me e ° @ e e s @s e ; e eo 2 rf : og oe 2 Poe ue Pe “ — . e (Loe *,@s A & 7 e ‘Pe @.% a6 e o bb e 1) . = ° ° @e%e° .@ ° ‘ e a e A e ® @e@* 6° 6 e pecs . e e® *@e-* 2 . ® ~ We rere e Ore) Se er \} e 4 3 ef e@ 2 9 2 Je ° é™ lee @e © 5 © 9! -2*s s ‘ee ws © 20 0,0 roe .o * % 0% | ° \j@% eo @ e en, o %e ee WY =e ? e,e@ @ t) ee! ee Q 2S — ° Za e@.erxre @ oe RAN i) aH G&S = © @, 04 & er, q he Et 626765 ee? & - @ re. * 4°? e ee%e ° rs ere »%.6@ . r ° @e@ @ee Ps SS Sa aN Fe . ®@aeg ee og ee VV al E e @eae e > 0:1 326 0-0). —* . = of e. 8" eo ee : eee 5 Een Meeete e@ @ee-. J ° e ® e? ® ee e @e»e? e ee. o &) : /\e@ ® ~9@ = / | ° : {7 We@iee . ~2 EF) eee , ° e overs @. e ° . 2 e @ e we @eo008 ° . ‘ . P» o.% 0 %e. ~! F ~ a a © @“ @ ~ a 8 1 ° ° bd f pe. ° . ad . . z fs 5a eae oa e 4 2 Fig. 1 Transverse section of muscle fibers from the pectoralis major of a rat which had been fed on fat meat. Fat droplets (black) are stained red by Herx- heimer’s Scharlach R. The larger fibers are ‘light fibers’, the ‘dark fibers’ are smaller. X 600. Fig. 2 Transverse section of muscle fibers from the pectoralis major of an emaciated rat. Fat droplets (black) are stained red by Herxheimer’s Scharlach R. Both ‘light and ‘dark’ fibers are shown. X 600. 10 H. HAYS BULLARD fat droplets. Bell (11) first clearly demonstrated that the fat content of muscle is largely dependent upon the nutritive condi- tion of the animal. This subject will be referred to later. The relative number of light, dark and intermediate fibers is known to be exceedingly variable. For any given muscle of a given species the percentage of each type is fairly constant under normal nutritive conditions, although individual variation occurs. Dark fibers have more interfibrillar substance or sarcoplasm and are commonly of lesser diameter than light fibers. However, the dark fibers in eye muscles (human) are as large or even larger than the light fibers. In the pectoralis major of the pigeon light fibers are exceptionally large with nuclei placed in the substance of the fiber, while dark fibers are small and the nuclei are peripher- ally situated. Mammalian skeletal muscle, in so far as I have observed, has peripherally situated nuclei in both types of fibers. The pectoral muscles of the bat are peculiar in that the fibers are all small and correspond to dark fibers as found in the pigeon. The fat content of the dark fibers of the bat as shown by Herx- heimer’s Scharlach R varies somewhat in the different fibers and this may be considered as an indication of the two types. The two types of fibers, dark and light, are clearly marked in the human fetus of seven months and of eight months. I have also found the two types in the ox fetus from 45 to 65 em. and at fullterm. In so far as I know dark fibers in the fetus have not been previously described. b. Relation of light and dark muscle fibers to white and red muscle Red muscles, as the pectoralis major of the pigeon and bat, commonly show a high percentage of dark fibers, while the white muscles of the rabbit and certain other animals may be made up largely or wholly of light fibers. By many text books and by even a recent author, Ewald (’10), the terms red and white muscle fibers are used synonymously with dark and light fibers. Such a terminology is probably founded upon a mise meeption and should be discontinued. The white muscles of fife frog during the winter season show under the microscope a irge percentage of very dark fibers and frequently red muscles, as cardiac muscle GRANULES AND FAT OF STRIATED MUSCLE 11 in certain individuals, show only light fibers. It is clear that dark fibers do not necessarily give a red color to muscle nor does the presence of light fibers in red muscle make it less red in appear- ance. According to Krause. (’11) red and white muscles in the rabbit differ only in number and arrangement of blood vessels and in amount of connective tissue. I am unable to say to what extent the red color of muscle is due to the presence of blood. Leliévre and Retterer (09) studied the structural differences between the red and white muscles of the rabbit. They concluded, among other things, that the membrane of Krause (Z, Strie d’ Amici) is absent from white muscle (adductor magnus). J have examined the fibers of the adductor magnus of the rabbit and find the mem- brane of Krause present and clearly visible in both fresh and fixed preparations. c. Morphology and position of fat droplets Krause (’73) observed a regular arrangement of the interstitial granules in transverse rows situated in segment J on either side of Krause’s membrane, Z. ‘The observations of Krause have been confirmed by Retzius (90), Arnold (00), Holmgren (’07,— ’10), and many others, and it has also been observed that the posi- tion of the granules as described by Krause applies to that of both the true interstitial granules and fat droplets. Retzius, Holmgren and others have described large interstitial granules as occurring in the anisotropic segment Q, (Briicke’s disc). The fat droplets of muscle fibers are in general spherical. Their form may be modified by the pressure of the muscle columns. Droplets that have a diameter exceeding one micron frequently show a certain amount of elongation in the direction of the longi- tudinal axis of the fiber. Exceptionally, the longitudinal diame- ter of elongated droplets is nearly twice the transverse diameter. Upon contraction of the fiber, droplets become more spherical or even flattened in the transverse direction. Occasionally, and_ especially in human muscle, the fat has a granular form, the evenly rounded contour of droplets being absent. Possibly this is due to post mortem changes. I am not here referring to the pigment ‘ 12 H. HAYS BULLARD granules of irregular form so frequently present in human muscle, especially in cardiac muscle. Fat droplets occur in varying sizes. Apparently the smallest droplets are beyond the limit of microscopic vision. Bell (11), staining muscle fibers by Herxheimer’s method, shows ‘liposomes’ (his fig.4, plate 16) which measure less than 0.5 mm. after a magni- fication of 1300. This means that they have a diameter of less than 0.5 wu. According to Heidenhain (’00), 0.2 » is the extreme lower limit of microscopical vision (n. a. |. 4), and if perchance any structure of smaller size were visible it would still appear to have a diameter of 0.2 micron. Herxheimer’s Scharlach R, as well as Nile blue frequently forms precipitates in the tissues but precipitate granules lack the refractive character of fat drop- lets and when of appreciable size the two are easily distinguishable. Precipitate in a finely granular form may be confused with minute fat droplets, and for this reason I have preferred to regard as probably a precipitate, all granules having an approximate diame- ter of 0.5 worless. The diameter of the fat droplets, which di ers with the nutritive condition and with the species of the animal, seldom, if ever, exceeds 3u in normal mammalian muscle. Fresh preparations stained in Herxheimer’s Scharlach R often show drop- lets as large as 5 to 6 », but the examination of fresh material to which no foreign fluids have had access has convinced me that such large globules arise by the confluence of smaller droplets. The fat droplets seldom show a regular arrangement within muscle fibers in preparations made from fresh material without fixation. 'To demonstrate the position of fat droplets, portions of muscle were stretched upon card board or small pieces of glass and while still warm placed in 20 per cent formalin in a 0.75 per cent solution of sodium chloride. After fixation of two to twenty- four hours, sections were cut on the freezing microtome and stain- ed by Herxheimer’s method or by the Nile blue method. Figure 3 shows the droplets in a portion of a longitudinal section of a dark muscle fiber from the pectoralis major of an adult cat. The droplets are in transverse rows in segment J on either side of the membrane of Krause, Z. An arrangement in longitudinal rows is also apparent. Figure 4 represents a portion of a light GRANULES AND FAT OF STRIATED MUSCLE 13 fiber from the same specimen. The droplets are fewer in number and somewhat smaller than in the dark fibers. The arrangement is similar in the two types. Droplets may be smaller than those shown in the figures and placed nearer to the membrane Z, thus making it difficult to see two distinct rows. Droplets in dark fibers which show a large quantity of fat, may all be of small size, not exceeding 1 and arranged as in figure 3. Dark fibers may show longitudinal rows of small droplets placed at frequent intervals between less frequent rows of larger, elongated droplets. In transverse sections the droplets are situated between the muscle columns or Cohnheim’s areas. The larger droplets are at nodal points in the sarcoplasmic net work. No droplets are found between the individual myofibrils within the muscle col- umns. In muscle fibers of a type which have large true intersti- tial granules (granules of segment Q, pectoral muscles of the pigeon and bat and in cardiac muscle), the fat droplets when small are placed in segment J, while larger droplets extend into segment Q. In this type of muscle the droplets in segment J may be placed on either side of the membrane of Krause, Z, but frequently drop- lets are in a single row occupying the position of the membrane. Fat droplets are also of almost constant occurrence in the sarco- plasm beneath the sarcolemma and surrounding the muscle fiber nuclei of skeletal muscle, and they are equally constant in the central perinuclear sarcoplasmic accumulations of cardiac muscle. d. Morphology and position of true interstitial granules Figure 5 represents the true granules in one of the dark muscle fibers of the pectoralis major of the pigeon, longitudinal section, Weigert method. The granules are rod shaped, being approxi- mately 1 » in diameter and 2 win length. In position they corre- spond to segment Q and represent Holmgren’s Q granules. Figure 6 represents a portion of a similar fiber, from the same muscle, stained with Nile blue sulphate after formalin fixation. By this method true interstitial granules are stained blue while fat droplets are colored red. The fat droplets, for the most part, are situated in segment J at the poles of the true interstitial gran- 14 H. HAYS BULLARD ules. Occasionally the substance of the Q granules appears to partially surround a fat droplet. In formalin-bichromate materi- al with the Weigert and Altmann methods, vacuoles left by the extraction of fat droplets during the paraffin process, are some- times seen within the substance of the granule but usually the vacuoles are in segment J at the poles of the granules as shown in figure 5. The vacuoles thus correspond in position to the polar fat granules which Holmgren describes as of occasional occurrence. Transverse sections show the true interstitial granules between the muscle columns. When demonstrated by the formalin-bi- chromate Weigert or Altmann methods, they appear rounded; or flattened in transverse section. In frozen sections stained with Cresylviolett or Nile blue sulphate, the granules are seen as stellate bodies which may occupy almost the entire space between the muscle columns (fig. 7). In describing this appearance in unstained preparations, KOélliker (’88) speaks of granules provided with wing shaped processes. When. frozen sections of formalin fixed material are treated with absolute alcohol and subsequently stained, Cresylviolett stains the true interstitial granules (pectoral muscles of the pigeon) rather faintly but the processes which give the stellate or irregular Fig. 3 Portion of a longitudinal section of a dark muscle fiber from the nor- mal pectoralis major of an adult cat. Fat droplets (black) are stained red by Herxheimer’s Scharlach R. Membrane of Krause is situated at Z; Q, position of anisotropic disc. > 1500. Fig. 4 Portion of a longitudinal section of a light muscle fiber from the normal pectoralis major of an adult cat, stained as in figure 3; Z, position of Krause’s membrane; Q, position of anisotropic dise. > 1500. Fig. 5 Portion of a longitudinal section of a dark muscle fiber from the normal pectoralis major of a pigeon. True interstitial granules, g, (black) are stained blue by a modified Weigert process. Fat droplets appear as vacuoles, a. The letter m indicates muscle columns; z, Krause’s membrane. X 1500. Fig. 6 Portion of a dark muscle fiber from the normal pectoralis major of a pigeon, stained by Nile blue. Fat droplets, a, are stained red (black); true inter- stitial granules, g, are stained blue (gray); m, muscle columns; z, Krauses’ mem- brane. X 1500. _ Fig. 7 Transverse section of a dark fiber and a portion of a light fiber from the normal pectoralis major of a white rat, stained with Cresylviolett. Fat droplets, a, are colorless or a faint red, true interstitial granules, g, are stained blue (black). The letter m indicates muscle columns. X 1500. Giie 15 GRANULES AND FAT OF STRIATED MUSCLE ~ o 7 5 = | | | | “4 poe als ee ee nlnelonteabon mil pr penton, 44 of 8 = 2.8 _ . Oi 6 aes BPO Wee gE Meine | | o > e e o. . ee aad ua mne! a2, . $0020 ax om mt podem Olen FES A coe ts eee FFL | 1 8 Gf memencnbms abmapmongacgme (O ; ate ~ OER Sean ey melee ®, ~ 2 ° ee so es. Festal Lar orshiha ta Gein Gee Piacoa : + poe 2 ee : Feige a etn ork en et a » . ‘4 POP b esse Pm hems ape Gow re, Cero t ~ ~yile ° Oo YS Fiat er ad ey ‘¥ i Py y s etn Wiest re ie N : bo) rae { o Oo < v re ¥.. 650 ' ! ' = g x os is ! nas Re hen 6 =e eo °° 98F gaice %2@ oe 49 20 6 et at kt toe! eer! ya OR Je : ¥ ec6 4 oe esalleeal oat ow OO (e} e * oe eo ee oe © od "a2 2 BSS ew mee / d A ° i er oe 3 ssen eee ome Move 2 vig x oe e200 00 se 00 00 2° 06 oe eo ee ‘ WP ere oc © 80 fe ww ee ve ob : fap) ae eee eee ee Te) ye 7) eo OF oe coer cere eo © one 9 ° oe lee rool com bal e ious jp Ose OF e ee Ped ee ad ee ¢0 eee ode deraibens emmy “4st ea VP Leen | ee e ee oe x e. o. © ee e . om a : = are a | Kee ». ° a ee ee eo #8 @ ® © 6 se «@ gee oe OP OD at me ee : ae » \ ee Psi i oa ‘ ! 0) = ) N 16 H. HAYS BULLARD appearance in transverse section are evidently not present. Nile blue still stains the granules with intensity but no stellate forms are visible. In this process, the fat droplets have been removed by the aleohol and also much of the substance which stains with Cresylviolett has apparently been extracted from the fiber or rendered incolorable. In formalin-bichromate material stained as either Weigert or Altmann preparations, paraffin sections, the granules seldom present the stellate form. The wing shaped pro- cesses of K6lliker may be dissolved during the process of paraffin embedding. Holmgren, however, has sometimes demonstrated the wing shaped processes by Benda’s mitochondrial method. The true granules were described above as having a rod shaped form in the breast muscle of the pigeon. This is not to be taken as invariably true. I have also observed in the pigeon muscle, dumb-bell and diplosomic forms in longitudinally cut fibers. At other times the shape is irregular and the granule is not wholly confined to the anisotropic segment. The longitudinal rows of granules in the light fibers of the pectoralis major of the pigeon are placed at greater intervals than in dark fibers though the gran- ules are somewhat smaller in the former. The true granules of the pectoral muscles of the bat are similar in number, size, and position to those of the pigeon. However, in the skeletal muscle of most mammals (dog, cat, rabbit) the gran- ules are of smaller size and fewer in number. In logitudinal see- tions they may be dumb-bell shaped, rodules, or slender thread structures, either confined to segment Q or extending through the entire distance between adjacent membranes of Krause, Z. They may occur as spherical bodies having a diameter of 1 u or less and situated in segment J on either side of Krause’s membrane. The appearance is then similar to that shown for fat droplets in figure 3. The occurrence of true granules in segment J is de- scribed by Holmgren (’10) as typical for mammalian skeletal muscle. The stellate or irregular forms in transverse sections shown by staining with Cresylviolett were of constant occurrence in all the animals used in this investigation. They may be demonstrated in both light and dark fibers, even when granules appear to be absent by the Weigert and Altmann methods. GRANULES AND FAT OF STRIATED MUSCLE ilivi In cardiac muscle (pigeon, dog, rat) the true interstitial granules are present in very striking numbers. They are usually confined to the segment Q and correspond to Holmgren’s Q granules. Regaud (’09) described and figured the granules in the cardiac muscle of the dog as plate-like structures, confined to segment Q and extending radially between the muscle columns from the periphery of the fiber toward the central sarcoplasmic column. I have examined the cardiac muscle of several dogs and find the true interstitial granules as described by Regaud. In the wing muscle fibers of insects the granules are very similar in form and position to either the granules of the pectoral muscle the pigeon and the bat or to those of cardiac muscle in verte- brates. The wing muscle fibers of the Belostoma Americana show rounded granules in transverse section similar to those of the pigeon. The fibers of dragon flies have granules of a plate- like form similar to those of the cardiac muscle of the dog. I have not examined fibers from the leg muscles of insects. Retzius (09) believed that the interstitial granules are united and held in position by a fibrous network, which he demonstrated with gold chloride. A comparison of gold chloride preparations with others made by the various methods already mentioned, leads me to believe that the appearance of a net work uniting the interstitial granules is to be interpreted as a precipitate of gold. The position assumed by the granules appears to be determined solely by their size and the pressure of the muscle columns. Per- haps also the position of both the true interstitial granules and fat droplets may be taken as affording evidence in support of the now commonly accepted view that the membranes of Krause are present in the sarcoplasm between the muscle columns. The dumb-bell and diplosomic granules may be formed by a thicken- ing of the muscle columns at Hensen’s line. After fixation, and due to the process required for embedding, the substance of the column shrinks leaving its impress upon the granules. The plate- like forms occur in types of muscle that present a radial arrange- ment of the muscle columns. The substance of the granule occupies the space between the columns and thus, in transverse sections, appears in the form of a plate. THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, No. 1 18 H. HAYS BULLARD In general it may be said that the shape of the true interstitial granules indicates that in fresh, unfixed muscle they are composed of a plastic, yielding substance which easily takes the form imposed by surrounding structures of a more resistant nature. Probably the term granule is a misnomer, but it is here used because it has become firmly fixed in the literature. KOolliker (88), Holmgren (07, 710), Thulin (09) and Knoche (09) believed that the true interstitial granules possess a limit- ing membrane. In Weigert, Altmann, Cresylviolett and Nile blue preparations, I have observed nothing which can be taken as indicating the existence of such a membrane. The membrane- like appearance in fresh unstained preparations is, in all probabil- ity, an. optical effect due to differences in refractive index. IV. GENERAL OCCURRENCE OF INTERSTITIAL GRANULES AND FAT DROPLETS General occurrence of true interstitial granules Kolliker (’89) described the true interstitial granules as of con- stant occurrence, sometimes in. enormous numbers, in the striated muscle fibers of all classes of vertebrates and insects. Knoll (91) observed the true interstitial granules in a large number of. animals including amphibians, reptiles, birds and mammals. Ret- zius (’09) described his sarcosomes (true interstitial granules) in insects and mammals. Altmann (’94) demonstrated the granules in insects andin the frog. Holmgren (’07—’10) described Kolliker’s granules as occurring in insects and in vertebrates, rabbit, guinea pig, rat and white mouse. The large true interstitial granules are included in nearly every description of insect muscle. Similar granules in vertebrate mus- cle, although described by the investigators just mentioned and by many others, have frequently been overlooked. This applies not only to text books but likewise to recent original articles. The white muscles of the rabbit show, by the Weigert or Alt- mann method, only a few granules or none at all and the red mus- cles may show but a limited number. In the dark fibers of the dog and the gray rat, granules are larger and somewhat more GRANULES AND FAT OF STRIATED MUSCLE 19 numerous than in the rabbit, especially in the muscles of the tongue and in the diaphragm. In the powerful and active pec- toralis major of the pigeon and of the bat, the true interstitial granules as demonstrated by the Weigert method are relatively large and occur in great numbers in each fiber (fig. 5). In so far as I have observed, the granules which stain blue with Cresyl- violett, (fig. 7), may be demonstrated in considerable numbers in every striated muscle fiber of all vertebrates. As already men- tioned, granules to be demonstrated by this method do not always correspond with those shown by the Weigert and Altmann methods. In cardiac muscle, as pointed out by Koélliker, Knoll and Holm- gren, the granules are especially abundant. I have examined the heart muscle of the dog, the rat and the pigeon. The num- ber and size of the granules is very striking. I have not been able to demonstrate the true interstitial granules in human cardiac or skeletal muscle, due doubtless to the fact that fresh material was not obtainable. General occurrence of fat droplets KOolliker (’88, ’89) describes fat droplets as of general occurrence in muscle fibers of insects and vertebrates. However, he was evidently somewhat in doubt as to their being true fat droplets. He speaks of the droplets as fat like granules or as the long known dark (fat?) granules. Walbaum (99) examined the muscles of 119 human bodies. He found fat droplets in some of the fibers of about two-thirds of the cases examined. Droplets were most numerous in the eye muscles and of very infrequent occurrence in the diaphragm. Ten per cent formalin was used as a fixative. He examined teased preparations in water and in normal saline and observed that many of the fatty droplets are left unstained by Sudan m1. Retzius (’91) believed that fat droplets are not normally present in muscle fibers. Among others Stadkewitch (94), Ricker and Ellenbeck (’99) and Kemp and Hall (’07), may be mentioned as failing to find fat droplets in the normal muscle fibers of adult vartebrates. As is well known, the muscle fibers of the winter frog are crowded with fat droplets while such drop- 20 H. HAYS BULLARD lets are usually supposed to be absent in summer frogs caught in the field. Fat droplets have often been overlooked in skeletal muscle due to the fact that they are so frequently lost in the fixatives employed (formalin) and may often be left unstained by osmic acid and Sudan ur. Bell (11) who employed Herxheimer’s Scharlach R on fresh tissue has demonstrated that ‘liposomes’ occur under normal conditions in all vertebrate muscle. He states that the number and size of the liposomes vary in different species and individuals and also with nutritive condition. He examined no human muscle. The dog, cat (figs. 2 and 3) and rat (figs. 1 and 2) may be men- tioned as examples of animals commonly having a large quantity of fat in their skeletal muscle fibers while the fibers of the ox and the rabbit have considerably less. I think that an extensive investigation might show that fibers of herbivorous animals do not store fat to such a great extent as is the case in carnivora. Human skeletal muscle. I have examined some of the muscles, usually diaphragm, pectoral and eye muscles from about twenty- five autopsies and conclude that fat droplets occur constantly and abundantly in normal human muscle. Fat in the diaphragm was present in large amount in about half the cases examined. In two or three cases the droplets in the fibers of this muscle were few in number or possibly absent but I think that this may be attributed to pathological conditions, poor nutrition. or to post mortem change. I have never failed to find fat in human eye muscle, usually in large amount. Cardiac muscle. I have examined sections from the right ven- tricle of the hearts of about twenty-five dogs and cats, a dozen rats and several mice. Fat in varying amounts was found within the muscle fibers of all these animals. In two dogs only a few small droplets were to be seen, but usually in this animal fat was present in moderate amount. An exceptionally large amount of fat was present in the cardiac muscle fibers of a well nourished rat and of a pregnant cat. Human cardiac muscle. Of fifteen hearts, fat droplets were present in ten. Two or three of the remaining five were examined GRANULES AND FAT OF STRIATED MUSCLE Pell after most decided post mortem changes had taken place. Fat droplets in the human cardiac fibers are commonly regarded as occurring only under pathological conditions. I believe that a thorough investigation of the subject, with the aid of the best technique would demonstrate that fat droplets of small size are of normal occurrence in human cardiac muscle fibers. Fetal muscle. Kainath (04) examined the skeletal muscles of the ox fetus. He found fine fat droplets in the fibers from the 3.5 to the 12.5 em. stage but none were present in the fetus of 20 em. and 40 em. Bell (09) found fat in the muscle fibers of the ox fetus from the 7 to the 28 em. stage, but observed none in the fibers of seven fetuses of later stages. The muscle fibers of the early fetus were not examined in this study but I have found a large amount of fat in the fibers of the ox fetus from the 35 cm. stage to full term. The dark fibers con- tain many fat droplets while light fibers have but a small number. I have also examined the skeletal muscles of a seven. months and eight months human fetus. The dark fibers were crowded with fat droplets. V. CHEMICAL NATURE OF INTERSTITIAL GRANULES AND FAT DROPLETS A qualitative chemical analysis, in vitro, of the true intersti- tial granules and fat droplets of muscle fibers is beset with obvious difficulties and such an investigation has never been attempted. One can, however, draw certain conclusions respect- ing the chemistry of these bodies by a consideration of the nature of the various methods used in demonstrating them in tissue sec- tions. a. Chemical nature of true interstitial granules Kolliker (’57) states that the true interstitial granules, being very pale, especially in mammalian muscle, have been overlooked by previous observers. He finds the granules insoluble in alcohol and ether. Kdlliker (’88) concludes that, chemically, the gran- ules are identical with no known substance. They contain no glycogen for they do not give the iodine reaction. Retzius (’90) oP H. HAYS BULLARD considers the true interstitial granules to be of a non-fatty nature. He terms them ‘sarcosomes’ in order to distinguish them from pathological fat droplets. Knoll (’80, 81, 91) believes the true interstitial granules of Kélliker to have a fatty marginal layer and a central portion possibly of lecithin. Arnold (’07) thinks that the glycogen. of striated muscle is bound to the sarcosomes. He observed that sarcosomes which contain glycogen stain by Best’s carmine method (’06) while those that are free from gly- cogen remain colorless. Regaud and Favre (’09) demonstrated eranules in the tongue muscles of the rabbit by Regaud’s formalin- bichromate iron-hematoxylin method. They believed these eranules to correspond to Koélliker’s granules. Chemically they were thought to be an albumino-lipoid. Bell(’11) finds that the large Q granules of insects contain no fatty substance and are widely different chemically from the interstitial granules of verte- brate muscle. He thinks that the microsomes of Altmann may be artefacts, and is evidently of the opinion that other observers have mistaken fat droplets in vertebrate muscle for the true inter- stitial granules. 1. Refractive character. Fibers from the pectoralis major of the pigeon or the wing muscles of an insect may be teased and placed, without the addition of fluid, upon a slide, the cover glass being applied with slight pressure. Such preparations show the fat droplets as highly-refractive globules but the true inter- stitial granules seem to have approximately the same refractive index as the substance of the muscle columns and are not clearly visible. However they may be seen as faintly-refractive bodies after normal saline has been drawn under the cover-glass. 2. Solubility. As has been observed by Ko6lliker and others the true interstitial granules are disintegrated and partially dis- solved by water. In order to test the effect of fat solvents upon the granules I have examined sections prepared by the paraffin process after fixation in 97 per cent alcohol. In sections from the heart or pectoral muscles of the pigeon, the granules in alcohol fixed material ‘are seen as broken fragments. A comparison of these sections with others made after formalin-bichromate fixation shows that a large part of the substance of the granules has dis- appeared from the alcohol fixed material. This suggests the idea GRANULES AND FAT OF STRIATED MUSCLE 20 that the partial disappearance of the granules from alcohol fixed material may be due to the solution of a fatty substance which can. be rendered insoluble by the action of potassium bichromate. It was found, however, that the granules of material fixed in 20 per cent formalin show no more shrinking after the paraffin pro- cess than do those of formalin fixed material which has been mor- danted in potassium bichromate before the alcohol and xylol pre- ceding embedding. Thin paraffin sections were also washed in several changes of hot ether four to six hours and subsequently examined under the microscope after staining with acid fuchsin or hematoxylin. The fat extraction by this method is considered more complete than by the Soxhlet’s apparatus as. ordinarily employed. Ether does not dissolve the true interstitial granules from paraffin sections of formalin fixed material taken from the pectoral muscles of the pigeon. The partial disappearance of the granules, from alcohol fixed material, takes place in the alcohol and xylol, and the subsequent treatment with ether appears to - have little effect. If we suppose that the shrinkage or disappear- ance of the granules in alcohol or xylol is due to the extraction of a fatty substance, it is also necessary to suppose that the fatty substance is in part rendered insoluble in alcohol, xylol and ether by the coagulative action. of the formalin on the non-fatty sub- stance of the granules. As will be seen below, however, staining with Cresylviolett indicates that the true interstitial granules are soluble in alcohol to a very considerable extent even in formalin fixed material 3. Results with Cresylviolett R R, Cresylechtviolett, and Nile blue sulphate. Krause (711) recommends Cresylviolett R B in dilute aqueous solution for demonstrating the interstitial granules in fresh tissue. I have used Cresylechtviolett and Cresylviolett R R which are apparently similar to the dye employed by Krause. The fat droplets are not stained to any considerable extent by Cresylviolett. Occasionally they show an exceedingly faint red color or a more intense peripheral blue staining but usually they ‘are left colorless. | As mentioned above, the true interstitial granules are par- tially dissolved by water. Since this is the case, one should 24 H. HAYS BULLARD not expect to obtain a true picture of the granules by the use of an aqueous staining solution on unfixed material. The arrange- ment of the granules is often very irregular in Cresylviolett prep- arations of unfixed material especially if the section is exposed to the action of water previous to staining or left too long in the stain, or if the material is taken from animals following rigor mortis. Under such circumstances granules are absent from por- tions of the fiber and are aggregated in masses within other por- tions of the fiber or beneath the sarcolemma. The blue stained substance, however, is not easily, if at all, soluble in water. Aque- ous solutions of Cresylviolett may also be applied to frozensections of material fixed fresh for two to twenty-four hours in 20 per cent formalin in a 0.75 per cent sodium chloride solution. Such prep- arations show a comparatively uniform arrangement of the blue staining granules corresponding to that of the true interstitial granules of Kolliker. It has already been mentioned that the wing shaped processes of Kolliker, which give the granules a stellate appearance, are not stained by Cresylviolett when the section has been previously treated with alcohol. In formalin fixed material the large gran- ules of the pectoral muscles of the pigeon can be stained intensely with Nile blue or faintly with Cresylviolett even after the action of alcohol. The true intersititial granules in the muscle fibers of the dog, cat, rat and rabbit stain with Cresylviolett and for the most part appear to be soluble in aleohol both in fresh material and in formalin fixed material for they cannot be stained when sections have been previously treated with absolute alcohol. When this material has been kept in 20 per cent formalin for sev- eral weeks, the wing-shaped processes and soluble granules seem to have disappeared and can no longer be demonstrated by Cresyl- violett or Nile blue sulphate. Even after prolonged exposure to formalin. the true interstitial granules in the pectoral muscles of the pigeon are still readily stained by Nile blue sulphate and faintly colored by Cresylviolett. If it be supposed that the alcohol-soluble substance of the true interstitial granules is a form of fat, the fact that it stains with basic dyes may indicate that it is a lipoid or fatty acid. GRANULES AND FAT OF STRIATED MUSCLE 25 4. Results with the methods of Weigert, Altmann, Benda and Regaud. 'To demonstrate the true interstitial granules, Altmann (94) employed his bichromate-osmic acid-fuchsin method. Holmgren (710) made use of Bend’s mitochondrial method and Regaud (’09) used his formalin-bichromate iron-hematoxylin method. I find that the granules may be demonstrated in a satisfactory manner by any of the above methods as well as by the Weigert method which involves formalin-bichromate fixa- tion followed by hematoxylin staining. Similar results by these methods is to be expected for the methods are chemically similar although the stains .employed, acid fuchsin, Crystallviolett, hematoxylin, and iron-hematoxylin, are of a varied character. Smith, Mair and Thorp (’08) have explained the chemistry of the Weigert hematoxylin process. They found that the method depends upon the oxidizing action of potassium bichromate upon unsaturated fats. The oxide of chromium forms with the fat molecules a compound which is insoluble in fat solvents and capable of forming a lake with hematoxylin. It is only during the process of oxidation that the fat-chrome compound forms the hematoxylin lake. After complete oxidation the staining no longer takes place. These observers found the method ap- plicable not only to unsaturated fats, as oleic acid and triolein, but also to lipoids in which unsaturated groupings occur such as cholesterin and cerebrosides. The work of Smith, Mair, and Thorp was confirmed and extended by Fauré-Fremiet, Mayer, and Schaeffer (10). They found that not only the unsatur- ated but also certain of the saturated fatty acids, mceluding palmitic, are rendered insoluble in alcohol and xylol by oxidizing reagents and also by the action of salts of the heavy metals. (Benda explained the action of the salts of copper on fatty acids as depending upon the formation of insoluble copper soaps.) These insolubilized fats were stained with more or less intensity by both acid and basic anilin dyes and in certain cases (after copper or chromic compounds, salts of iron and of zinc) a hematoxylin lake was formed. The phosphatid lipoids were not rendered insoluble in xylol by the action of salts of the heavy metals, but were insoluble after chromic and certain other 26 H. HAYS BULLARD oxidizing compounds. Lipoids rendered insoluble by chromic compounds stained with considerable intensity by Orange G but could be stained with the anilin dyes only when the potassium bichromate had been kept warm during the process of oxidation. The hematoxylin lake in the case of the lipoids, did not follow excepting after a mordant such asiron alum. It was also observed that both albumino-lipoids (lecithalbumin) and mixtures of fatty acid and albuminoids were precipitated by formalin in such a way as to render the fatty substances practically insoluble in ordinary fat solvents. For example, oleic acid in a precipitated albuminous mixture was stained by various methods, even after the action of alcohol and alcohol-ether for several days at a temperature of 85°C. The methods of Altmann, Benda and Weigert, although variously modified are, according to these observers, based on. the same chemical principles and give almost identical results when applied to the mitochondria (Altmann’s granules or the true interstitial granules of this paper). After an extended inquiry into the chemistry of the mitochondria, they conclude that the granules contain a fatty body which is neither a neutral fat nor a soap but is probably an unsaturated fatty acid, absorbed by an albuminous granule or present in an albumino- lipoid compound. If we assume with the authors just quoted and with Regaud . and Favre (’09) that the true interstitial granules are an albumino- lipoid or a fatty-acid albuminous mixture, the action of formalin in. partially protecting them against fat solvents is explained in that the albuminous component is coagulated by the formalin and the fatty component is thus rendered less easily extractable. ‘The same assumption also permits us to explain the action upon the granules of the methods of Altmann, Benda, Weigert and Regaud. It would, of course, be a mistake to consider these methods as specific for albumino-lipoids. They are of wide application and do not afford distinctive evidence as to the chem- ical nature of the substances stained. 5. Results with acid fuchsin. Knoche (’09) obtained a micro- chemical xanthoproteic reaction with the true interstitial granules of K6lliker and belived that the proteid thus shown was an GRANULES AND FAT OF STRIATED MUSCLE ah albuminous substance. He states that the granules have a cap- sule which stains with acid fuchsin but he does not give the details of his technique. As has been previously mentioned, the entire granule is readily stained in formalin-bichromate material with Altmann’s acid fuchsin. Employing material from the pectoral muscles of the pigeon and bat and from the wing muscles of in- sects, I have also found that Altmann’s acid fuchsin stains the granules after simple formalin saline fixation (paraffin process) the potassium bichromate being unnecessary. The fragmented granules in. alcohol-fixed material are not stained by Altmann’s acid fuchsin. , Smith and Mair (11) find that lecithin and sphingosine stain readily with acid fuchsin both before and after the action of potas- sium bichromate. They think the presence of either of these substances would explain the staining of Altmann’s acid fuchsin granules. Pure lecithin, according to these observers, does not stain by the Weigert process, but they add that it stains readily if it has the slightest admixture of cholesterin. Fauré-Fremiet, Mayer and Schaeffer (10) state that lecithin and other lipoids fail to stain by the Weigert process, but may be stained by hema- toxylin if preceded by iron alum. Since the true interstitial granules stain readily by the Weigert process, the iron alum not being necessary, we may conclude that if the staining of these granules depends largely on lecithin, as suggested by the acid fuchsin. method, the lecithin is not in a pure state. Fatty acids, according to Smith and Mair (711), do not stain with acid fuchsin either before or after the action of potassium bichromate. Fauré-Fremiet, Mayer and Schaeffer (10), on the other hand, find that fatty acids are faintly stained by this dye, both before and after bichromating, presumably with greater intensity in the latter case, for they think that the presence of fatty acid would account for the staining of Altmann’s granules. I have stained tissue paper smears of oleic acid (Kahlbaum) after treatment for a variable length of time (one to six days) in. satu- rated potassium bichromate. The smears were stained with acid fuchsin either Altmann’s mixture or in. alcoholic solution, heating according to:the method of Altmann. The droplets were stained 28 H. HAYS BULLARD a somewhat pale red. The color in the case of oleic acid appears to be too faint to fully account for the intense red of the true interstitial granules, and thus it is doubtful that these granules contain a pure Oleic acid. 6. Results with Sudan III, osmium tetroxide, gold chloride. Sudan mr does not color the true interstitial granules unless we take into account an extremely faint yellow to be obtained after the action of potassium bichromate. ' The granules are slightly darkened, but not blackened, by 2 per cent osmic acid followed by pyroligneous acid or by alcohol for reducing the osmium. Retzius (90), Knoll (91), and others have stained the true interstitial granules’ with gold chloride but the gold precipitate is not con- sidered differential for the presence or absence of fat. 7. Summary. The observations presented above are of too general a character to permit of definite conclusions as to the chemical nature of the true interstitial granules of Koélhker. It is certain that the granules contain a non-fatty element, probably of a proteid nature. It may be stated that the substance upon which depends their staining by basic dyes, as well as by the more complex methods of Altmann, Weigert and Regaud, is a substance soluble in fat solvents. In part this soluble substance may be protected from fat solvents by the action of formalin as well as by chrom-osmic mixtures. The solubility and staining reactions of the granules indicates that they may be an albumino-fatty compound or mixture, possibly an albumino-lipoid. There is no reason to suppose that the granules in muscle fibers are funda- mentally different chemically from granules to be demonstrated by similar methods in other tissues of the body. It is reasonable to suppose that the true interstitial granules of muscle fibers are subject to some variation chemically in different species and under varying nutritive conditions. b. Chemical nature of fat droplets The fat droplets of muscle fibers are mentioned by many obser- vers but few have attempted to determine the exact chemical nature of the fat. Usually, it seems, the droplets have been looked GRANULES AND FAT OF STRIATED MUSCLE 29 upon as neutral fat. As already mentioned, Knoll (’80, ’81, ’90) thought the true interstitial granules to be composed, in part at least, of lecithin, but he too considered the fat droplets to be neutral fat. Bell (10) holds that neutral fat droplets in muscle fibers are readily stained by simple alcoholic solutions of Scharlach R, but many ‘liposomes’, which are not so highly refractive as neutral fat droplets and consist wholly or in part of lipoids, can be stained only by alkaline Scharlach R (Herxheimer’s method). Bell (11) thinks the liposoms consist mainly of olein together with some low-melting fat other than olein. He states that many faintly-refractive liposomes which do not stain readily with simple alcoholic solutions of Scharlach R or with osmic acid, are stained somewhat faintly by Herxheimer’s method and are composed in part of a substance other than fat, possibly an albu- mino-lipoid. Liposomes which stain faintly by Herxheimer’s method and contain a non-fatty element are believed by Bell to be of most common occurrence in the muscle fibers of poorly nourished individuals. 1. Refractive character: Double refraction. The fatty droplets of muscle fibers may be seen in fresh tissue to which no foreign substance has had access. Preparations are made by rapidly teasing the fibers on a slightly warmed slide and applying a cover glass with slight pressure. The droplets present the highly refractive appearance characterisitic of fat droplets and must be regarded as pre-existing bodies, that is to say they are not produced by histological reagents. Fat droplets are well brought out in fresh preparations mounted in normal saline. They vary some- what in refractive index in different individuals: but usually the variation in a single preparation is not pronounced. The true interstitial granules may also be observed in such preparations. These granules likewise vary somewhat in refragtive index but are usually less refractive than the fat droplets. Judging merely from refractive index certain granules may be classed as either faintly refractive fat droplets or highly-refractive true interstitial granules. Preparations mounted in 2 to 5 per cent potassium hydroxide show the fat droplets very clearly for an hour or more but the true granules disappear almost immediately. 30 H. HAYS BULLARD Bell (11) states that the fat droplets of muscle fibers are all isotropic. I have examined only afew specimens with the micro- polariscope. The droplets were always singly and not doubly refractive. This shows that the droplets are not a fat which is fluid crystalline in form, such as the cholesterine compounds. 2. Solubility. The fat droplets of muscle fibers are readily soluble in cold absolute alcohol and in ether. Ninety-five per cent alcohol usually dissolves the droplets from frozen section or teased preparations in a few minutes. ‘Tissue fixed in seventy per cent alcohol frequently shows a gradual diminution of the quantity of fat. It is well recognized that tests of solubility are of little value in determining the chemical character of fats in the tissues, especially as such fats, at least in most cases, are not in a pure state but exist as mixtures. Neutral fat has usually been considered insoluble in 70 per cent aleohol. However the fat droplets of muscle fibers, having a diameter of but 1 to 3 un, must be regraded as in'an extremely fine state of division, thus favoring prompt solution and, moreover, the quantity of solvent is very many times that of the fat dissolved. The fact that fat droplets in muscle fibers are sometimes dissolved by 70 per cent alcohol does not prove that they are not neutral fat. 3. Results with Scharlach R and Sudan III. Bell (10) states that I had shown clearly the great superiority of alkaline alco- holic solutions of Scharlach R and mentions that my results had not yet been published. My observations concerning the stain- ing of fat droplets in muscle fibers with alkaline alcoholic solutions of Scharlach R and Sudan ur and with simple alcoholic solutions of the same dyes, were made in the Laboratory of Anatomy of the University of Missouri three years ago and are here given ina corrected form. At that time I observed the position of fat drop- lets in muscle fibers, a subject already discussed in this paper. Schlarlach R and Sudan 1 are usually employed as saturated solutions in 70 to 80 per cent alcohol. Such solutions frequently fail to stain the fat droplets of muscle fibers. The best results are obtained by heating the alcohol at the time of preparation of the stains or by permitting a certain amount of evaporation during the staining process. Even after an application of twenty- GRANULES AND FAT OF STRIATED MUSCLE dl four hours, these stains may color only a small part of the total number of fat droplets which can be seen in fresh preparations or demonstrated with Nile blue. Herxheimer (’04) does not state specifically that bis alkaline-alcoholic solution of Scharlach R will color any fat droplets which cannot be stained with simple aleoholic solutions but he quotes Erdheim (’03) as having made such a claim. In so far as I have observed, alkaline-alcoholic solutions of Scharlach R and Sudan 1 stain all the fat droplets of muscle fibers. Herxheimer’s stain usually gives a deep red color to droplets faintly stained or left colorless by simple alcoholic solutions. As already stated, true interstitial granules are not stained by Sudan m1 and Scharlach R. Frozen sections or teased preparations of muscle fibers, as well as of other tissues, which, when stained by the ordinary stock solutions of Sudan 1 and Scharlach R in 70 per cent alcohol may appear fat free, are some- times shown to be crowded with fat droplets when examined in the unstained condition or when stained with Herxheimer’s stain, or with Nile blue followed by immersion in an alkaline medium. In some specimens of muscle the simple alcoholic solutions stain all the fat droplets which can be seen in the fresh tissue. The fact that fat droplets in muscle fibers are frequently left unstained by the less concentrated solutions of Sudan mr and Scharlach R does not seem to offer sufficient proof that such drop- lets are not neutral fat. Fat in adipose tissue of mammals which presumably is neutral fat, is occasionally colored so faintly by these stains that were it in finely divided droplets it would be almost colorless. I do not share the belief advanced by Bell that we must suppose the droplets to contain an admixture of albumin or other non-fatty substance. The droplets in the muscle fibers of emaciated individuals, in so far as I have observed, stain with as great intensity by Herxheimer’s method as do those of well nourished individuals. The intensity with which droplets are stained both with Herxheimer’s stain.and with simple alcohol- lic solutions of Scharlach R depends as much upon the conditions under which the dye is used as upon the nature of the fat. The fact that droplets stain faintly cannot in itself be taken as suffi- cient proof that they contain a non-fatty element. 32 H. HAYS BULLARD 4. Osmium tetroxide. It is well known that osmic acid is reduced only by the unsaturated fats, the reduction depending upon the oxidation of the fat. Therefore it has been considered that fat droplets that are blackened by osmic acid consist, wholly or in part, of unsaturated fats. However, unsaturated fats present in small amounts as mixtures with saturated fats may fail to blacken with osmic. The fat droplets in muscle fibers of many animals do not reduce osmic acid. While in certain individuals the fat droplets blaken with osmic acid, in other individuals of the same species the reduction does not occur. This shows that the unsat- urated fat in the droplets is variable in amount. : 5. Results with the Nile blue method. Smith (07) explained the staining of fats with the basic anilin dyes as depending upon the formation of color-soaps by the action of fatty acids and color bases. Neutral fat, as such, can not be stained by basic anilin dyes, but after hydrolysis the free fatty acid combines with the color base. This observer also found that neutral fat in the tis- sues is in a very unstable condition, being hydrolyzed by dilute acids and even by the carbon dioxide of the air. Thus neutral fat droplets in stained sections, after having been hydrolyzed into fatty acid and glycerine by exposure to the air were observed to take the color of the basic stain. Smith found that Nile blue sulphate initially gives a red color to both neutral fat and fatty acid. Neutral fat retains the red staining quality but subse- quently fatty acids form color-soaps with the Nile blue base, the deep blue of the soap obscuring the comparatively faint red color of the fatty acid. Aschoff (09) found that the phosphatid lipoids and cerebroside are also colored blue by Nile blue sulphate. McCrae and Klotz (10), who used Nile blue on sections of fatty liver, state that they experienced some difficulty in interpreting their results. According to Klotz (09) ‘‘The blue coloration obtained in staining sections with Nile blue is not to be depended upon. as indicating in every instance the presence of fatty acids, as the shade of the color is influenced by external circumstances. ”’ He, however, does not specifically state by what circumstances the color is influenced. Nile blue has been used as a means of investigating the chemical nature of fat droplets in tissue sections, but in so far as I know GRANULES AND FAT OF STRIATED MUSCLE 33 the method has not been applied to muscle fibers. I have fol- lowed directions given by Smith and Mair (711) for the use of Nile blue sulphate but the results have not been very satisfactory. Apparently the fat droplets are decolorized by the 2 per cent acetic acid used as a differentiating fluid. The method which I have used with best success is given here under the heading ‘‘ Material and methods.’’ In brief it consists in setting free the color base by the addition of a small amount of alkali to the medium in which the section is mounted, or by washing in slightly alkaline water after differentiating in distilled water. Precipitates are formed if sections are not carefully washed before being placed in the alkaline solution. With a little care in making the prepara- tions with Nile blue chlorhydrate, and apparently with the sul- phate also, it is possible to stain all the fat droplets to be found in muscle fibers. That is to say, Nile blue will stain droplets that are not stained by simple alcoholic solutions of Sudan m1 and Scharlach R or by osmic acid. With Nile blue, as with other stains, fresh tissue should be used if all the droplets are to be stained, for they frequently disappear in fixed tissue. As has already been said, Nile blue sulphate stains not only the fat droplets but, in formalin fixed material at least, it also stains the true interstitial granules. This is best shown by staining a section from the pectoral muscle of a pigeon or bat. The true interstitial granules are especially abundant and stain a bright blue, the shade depending upon the length of time in the stain and upon the reaction of the mounting medium. The fat droplets which may also be present in large numbers, usually stain a some- what faint red but sometimes in various shades of purple or blue. In formalin fixed specimens of material containing large true inter- stitial granules, it is not difficult to distinguish the latter from fat droplets since the granules do not have the globular form of the droplets and for the most part present color differences. The color assumed by the fat droplets when stained with Nile blue is of some importance as it may help in identifying the fats chemically. The color of the droplets depends, to a considerable extent upon the time occupied in staining and upon the alkalinity of the solution to which the stained section is exposed, as well as THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, No. 1 34 H. HAYS BULLARD upon the length of time left in this second solution. The red staining takes place rapidly. The blue staining is sometimes well marked after fifteen minutes but may not appear for several hours or may not occur at all. The color is also influenced by the reac- tion of the mounting medium. Jn any alkaline medium the pale blue droplets tend to become red, while in an acid medium the red droplets tend to become blue. The entire mass of droplets may stain uniformly either red, blue, or in various shades of purple. On the other hand, a droplet colored red may include within its mass one or several clearly marked smaller droplets which stain an intense blue. Again, the periphery of a droplet may show blue staining while the center is red. After preparations have been mounted for a variable number of days, droplets which at first were red may assume an intense blue color. In other prepa- rations the red color is retained for months. I have observed a certain amount of blue staining of the fat droplets in every species of animal from which material was taken, including man. The blue staining compound is found in muscle fibers taken from well nourished animals as well as in fibers from animals poorly nour- ished. It is difficult to interpret the color reactions of Nile blue upon the fatty substances of muscle fibers. The blue color of fat drop- lets may possibly indicate that they are, in part, either fatty acid or a lipoid substance, while the red color indicates the presence of neutral fats. However, the blue color of the fat droplets may, as we have seen, indicate the presence of a neutral fat which is easily hydrolyzed, the fatty acid then forming a color soap. When the blue staining takes place only after sections have been mounted several days, it is, in all probability, dependent upon the gradual hydrolysis of neutral fat. With fresh tissues treated with the dye for a few minutes only, the blue staining, which is sometimes immediately apparent, may be due to similar changes in the neutral fat. It is possible that blue staining of fat droplets in fresb unfixed tissue or in tissue obtained some time after the death of the animal, may be due, in part, to the solution in the fat droplets of the blue staining substance of the true interstitial granules. It was pointed out by Smith (’07) that fat droplets in GRANULES AND FAT OF STRIATED MUSCLE 30 tissues may be hydrolyzed by the action of the formalin fixatives. It follows that in formalin fixed material the blue staining with Nile blue is often an expression of neutral fat which has under- gone hydrolysis. The fat droplets of the muscle fibers of many individuals of a species show no blue staining after a prolonged exposure to the stain. From this we may conclude that the droplets in these individuals contain little or no free fatty acids, phosphatid lipoids, or cerebroside. In no case, in so far as I have observed, does the staining with Nile blue afford convincing proof that any sub- stance other than neutral fat is normally present in the fat drop- lets of muscle fibers during the life of the animal. Contrary indigations may be due to postmortem changes. 6. Results with the methods of Benda, Fischler and Klotz for free fatty acids and soaps. According to Benda, neutral copper acetate forms, with free fatty acids, colored copper soaps which for the most part are insoluble in fat solvents. The methods of Benda, Fischler and Klotz depend upon this reaction. Fischler (’04) found that the fatty acid copper compound forms a lake with hematoxylin. He also stained soaps in the tissues by the same methods, the soluble potassium and sodium soaps being first converted into insoluble calcium soaps by the action of calcium salicylicum. Klotz (06) suggested further modifications. Bell (11) used the above methods on preparations from a considerable number of muscles, but in no case was able to get the color reac- tion for free fatty acids or soaps. He concludes that the lipo- somes (fat droplets) of muscle fibers do not contain either fatty acids or soaps. I have used these methods only to a limited extent and my results are in agreement with those of Bell. However a positive result with the methods of Fischler and Klotz should not be taken as certain proof of the existence of free fatty acid during life. The fixative employed in both these methods contains formalin and in the method of Klotz, acetic acid is added. It is thus possible that the staining occasionally depends upon the previous hydrolysis of neutral fat. 7. Results with the Weigert method and related methods. We have already discussed these methods and seen that they give 36 H. HAYS BULLARD positive results with the true interstitial granules. The fat drop- lets of muscle fibers are not easily rendered insoluble by the action of chromic compounds. For the most part the droplets are dissolved by the alcohol or xylol used in the paraffin process even when the tissues have been treated with 10 per cent potas- sium bichromate for several days at 37°C. It follows then that the fat droplets in muscle fibers are not shown by the Weigert or iron-hematoxylin methods as ordinarily employed. When chrom-osmic fixatives are used, as in the methods of Altmann and Benda, the droplets are sometimes blackened and rendered insoluble, but usually they are still soluble. When, as rarely happens, fat droplets are rendered insoluble by the bichromate- osmic mixtures, and at the same time not blackened by osmie, they may be stained by Benda’s iron-alum Krystallviolett and probably also by hematoxylin or iron-hematoxylin. Since the investigations of Smith and Mair (’08, 710, ’11), Aschoff (’09), and of Fauré-Fremiet, Mayer and Schaeffer (10) have shown that the phosphatid lipoids, combinations of cholesterin and fatty acids, as well as cerebroside, are rendered insoluble by the action of potassium bichromate, we may conclude that the fat droplets of muscle fibers do not, to any very considerable extent, consist of these fats. Saturated neutral fats are not rendered insoluble by the action of potassium bichromate and triolein is acted upon only very slowly. The fat droplets of muscle fibers not being readily acted upon by potassium bichromate, react as if they were composed wholly or largely of neutral fat. 8. Formalin fixation. Bell (10) pointed out the fact that fat droplets in muscle fibers and other tissues are frequently not preserved by formalin fixation. Droplets of ordinary neutral fat he states are not affected in their staining by formalin fixation, but many faintly-refractive fat droplets, consisting wholly or in part of lipoids, cannot be stained after formalin fixation. The faintly-refractive droplets are either removed or rendered invisible by fixation. He finds that the action of the formalin fixative in _ one tissue may be unappreciable for weeks and in another nearly all the liposomes may be removed in a few minutes. He states that the varying effect of the fixative is probably due to the vary- GRANULES AND FAT OF STRIATED MUSCLE Bh ing chemical composition of the liposomes. Bell also found that usually the fat droplets in the muscle fibers of adult and well ' nourished animals are less affected by formalin than are the drop- lets of young and poorly nourished animals. However, many exceptions to this rule were mentioned. Bell (’11) confirms his former observations and states further that the faintly-refractive liposomes, which are removed by formalin fixation, consist in part of a substance other than fat, possibly an albumino-lipoid. He believes that the disappearance of the liposomes is probably due to autolysis. I have repeatedly observed the gradual disappearance of fat droplets from tissue fixed in formalin. However, I am not certain that neutral fat is not affected by formalin fixation. Smith (11) in fact has found that ordinary formalin solutions hydrolyze the fat droplets of frozen sections which are kept in the fixative. I have frequently but not invariably found that the blue staining of fat droplets with Nile blue is more marked in formalin fixed material. Free fatty acids are soluble in Herxheimer’s staining solution and hence cannot be demonstrated by Herxheimer’s method. The fat droplets of formalin fixed material frequently disappear, not being visible either as crystals or as droplets stain- ing blue with Nile blue. This indicates that the final change which takes place in the fat is not be to explained on the basis of simple hydrolysis. I believe, however, that the initial change may be hydrolysis of an unstable neutral fat. The fat droplets of the muscle fibers of the ox fetus, for the most part, stain red with Nile blue but after several days the droplets in the stained section assume a blue color. This change of color indicates that the fat is in an unstable condition and can be readily hydrolyzed. The fat droplets of the fetal tissue were not permanently preserved by formalin fixation. The observations of Bell and Smith, as well as my own, are based on the use of ordinary commercial formalin. This solution contains variable quantities of formic acid and possibly acetic acid. The hydrolysis of the fat may depend upon the presence of these impurities. It is thus possible that the variable action of formalin is to be explained in part by the variable chemical composition of the fixative. 38 H. HAYS BULLARD The action of formalin on the fat droplets is not what we should expect if the droplets were an albumino-lipoid as suggested by Bell. In fresh unfixed muscle fibers the droplets readily coalesce to form larger globules. In formalin fixed material the coales- cence is not so apparent, for the substance immediately surround- ing each droplet has been coagulated. However by the examina- tion of carefully teased muscle fibers it is easy to convince oneself that the droplets themselves are not hardened but may still coalesce. If albumin were present to any considerable extent, the droplets would be fixed and no coalescence would take place. Fauré-Fremiet and his collaborators have shown that albumino- lipoids are coagulated by formalin in such a manner as to render the fatty substance almost insoluble in fat solvents. The fat droplets of formalin fixed muscle fibers are apparently as easily dissolved by alcohol as in fresh tissue. 9. Summary. The fat droplets of muscle fibers are not largely composed of fatty acids or soaps for they do not stain by the methods of Benda, Fischler and Klotz. They are not fatty acids for they do not stain readily with basic anilin dyes. The fat droplets do not contain cholesterin esters to a very considerable extent for they do not present the characteristic anisotropic fluid-crystalline form. The droplets are not phosphatid lipoids or cerebroside for these substances are easily rendered insoluble by potassium bichromate, whereas the fat droplets of muscle fibers are not rendered insoluble. Also phosphatid lipoids and cerebroside stain with basic dyes, giving blue with Nile blue, while the fat droplets of muscle, at least for the most part, stain red with this dye and are colored blue only after a chemical change has occurred in the fat. Fat droplets in muscle fibers are readily stained by alkaline-alcoholic solutions of Scharlach R and Sudan 1 but are frequently left unstained by simple alcoholic solutions of these dyes. The evidence which tends to show that the fat droplets of muscle fibers are neutral fat (glycerin esters of fatty acids) is largely of a negative character. It is improbable that they are pure neutral fat, yet it may be said that no certain proof has yet been offered that any substance other than neutral fat is present in the fat droplets of muscle fibers during the life of the animal. GRANULES AND FAT OF STRIATED MUSCLE 39 VI. PHYSIOLOGICAL SIGNIFICANCE OF INTERSTITIAL GRANULES AND FAT DROPLETS Physiological significance of true interstitial granules Several investigators have held that true interstitial granules give origin to fat droplets either by a fatty metamorphosis or by serving as a focus about which fat is deposited. Kolliker (88-89) belived that fat droplets in muscle fibers arise from true inter- stitial granules by a process of fatty metamorphosis and Schaeffer (93) advanced similar views. Holmgren (10) states that the deposition of fat in muscle fibers is apparently influenced by the true interstitial granules but he thinks the granules are actually transformed into fat only under exceptional or pathological con- ditions. Altmann (’94) held that his bioblasts (true interstitial eranules) are not transformed in toto into fat but act as a focus within and around which fat is accumulated. Arnold has advo- cated similar views. Bell (’11) does not believe that granules which stain with acid fuchsin (Altmann’s granules) give origin to fat and he is of the opinion that true interstitial granules do not occur in vertebrate muscle. His conception of the deposition of fat in muscle fibers is nevertheless essentially in accord with the theory advanced by Altmann. Bell holds, namely, that the fat of muscle fibers is deposited as ‘liposomes’ around a pre-existing non-fatty body, possibly an albumino-lipoid. As we have seen, Altmann’s granules are probably an albumino-lipoid formation and conversely albumino-lipoids may be expected to stain with Altmann’s acid fuchsin. The reader is referred to the literature for a presentation of the arguments offered by various authors im support of the idea that true interstitial granules give origin to fat droplets or serve as foci about which fat is deposited. It may be said that, at present, the truth of such a conception is not sufficiently demon- strated to warrant us in believing that there is a genetic relation- ship between true interstitial granules and fat droplets. According to Holmgren (’07, 10) the colorable substance (method of Benda) of the interstitial granules is necessary to the proper functioning of the contractile elements. During the 40 H. HAYS BULLARD latent period, substance from the granules was thought to pass into the muscle columns there to be used in the stage of active contraction. The examination of insect and vertebrate muscle fibers, by the various methods employed in this study, has afforded little in support of Holmgren’s views concerning the physiologi- cal significance of the granules. Knoll (’80) and Knoll and Hauer (’92) found that the true interstitial granules are not removed in inanition. Feeding experiments with a dozen white rats lead me to conclude that the alcohol-soluble portion of the true interstitial granules which stains with Cresylviolett is increased in amount when rats are heavily fed and decreased when the animals are kept on low rations. This may indicate that the alcohol-soluble substance is of metabolic importance and such an assumption would seem the reasonable one. Before dismissing the subject of the physiological significance of true interstitial granules, it should be mentioned that Arnold (09) believes that glycogen is bound to the sarcosomes (true interstitial granules), and. Kingsbury (’12) thinks that mitochon- dria, in general, act as a reducing agent and in certain cases may be concerned in exercising the function of cell respiration. Physiological significance of fat droplets The presence of fat droplets in striated muscle fibers has been regarded by Van Gehuchten (’89) and others, as of pathological significance. Probably this is still: the prevailing opinion with respect to cardiac muscle. Schaeffer (93) believed that fat drop- lets in the skeletal muscle fibers of vertebrates may occur under normal conditions but are usually pathological. Walbaum (’99) found that fat droplets were of very frequent occurrence in normal human muscle fibers but thought the quantity of fat bore no direct relation to the nutritive condition of the individual. Kél- liker (88) regarded the fat droplets of insect muscle as reserve food material. Knoll and Hauer (’92) found: that fat droplets in the muscle fibers of pigeons are removed by starvation but the true interstitial granules are not removed. Krause (11) states that the fat droplets in muscle fibers are not independent of the GRANULES AND FAT OF STRIATED MUSCLE 41. nutritive condition of the animal. Bell (’11) found that the liposomes (fat droplets) of the striated muscle fibers of rats were entirely removed when the animal was starved until it had lost 25 per cent or more in body weight. During starvation, the lipo-_ somes gradually became faintly refractive and decreased in size, number and in staining intensity with Herxheimer’s Scharlach R and with osmic acid. When the starved animal was again given food the liposomes gradually reappeared, increasing in size, number, refractive power, and staining intensity as the animal gained weight. In normal rats which were fed on fat meat for several days, the liposomes were greatly increased in number, size, and staining intensity. When summer frogs were fed heavily on olive oil or fat meat, there was a striking increase in size, num- ber and staining intensity of the liposomes. No changes were produced in the liposomes by the feeding of grape sugar, starch, palmitic acid, sodium oleate, or lean meat. Since the liposomes stained faintly when they first appeared, Bell supposed that they then contained a relatively small percentage of fat together with some substance other than fat, possibly an albumino-lipoid. He regarded the liposomes as foci where fatis deposited and concluded that they consist of reserve food substances mainly, at least, in the form of fats. ; In this connection I have examined the muscle fibers of a dozen white rats on various nutritive planes. Figure 1 represents fibers from the pectoralis major of an adult rat which had been heavily fed on fat meat for seven days. The fat droplets were stained with Herxheimer’s Scharlach R. Figure 2 shows muscle fibers from the pectoralis major of a rat which had been kept for ten days on short rations of a fat free diet consisting mainly of cellulose. Fat cells were almost completely absent from the sub- cutaneous tissue and mesentery of this animal. Fat droplets are practically absent from the light fibers as shown in the figure, while dark fibers have a smaller quantity of fat than is normally present even in light fibers. In the diaphragm of this animal, the quantity of fat, although greatly reduced from the normal, was somewhat greater than that found in the pectoralis major. The amount of fat in the cardiac muscle fibers of emaciated rats was less than in fibers of well nourished individuals. Animals 42 H. HAYS BULLARD fed on fat meat showed an increased amount of fat in the cardiac fibers but the increase was not so great as in skeletal muscle. The muscle fibers of the pectoralis major of normal rats kept on a diet of bread and lean meat show fat intermediate in amount to that represented in figures 1 and 2. In several rats the superficial fibers of the pectoralis major contained less fat than those some- what removed from the surface. The fibers illustrated in the figures were not superficially placed. After rats have been fed, on fat meat for a few days, the quantity of fat in the muscle fibers appears to have been increased to the maximum. Further feeding increases the amount of connective tissue fat but seems to have no effect upon the fat in the muscle fibers. As already mentioned, dark muscle fibers are more clearly marked in well nourished animals than in emaciated animals of the same species. The fat droplets of muscle fibers are clearly to be regarded as reserve foodstuff. The work of Bell in this respect is so con- vincing as scarcely to require confirmation. I have not made observations on the effect of starch, sugar or protein diets, as has Bell. VII. SUMMARY AND CONCLUSIONS The interstitial granules of striated muscle may be classed as true interstitial granules and fat droplets as was done by Kolliker. Both the granules and fat droplets are factors in bringing about the dark or cloudy appearance of muscle fibers. The two types of fibers, dark and light, the occurrence of which is well known in adult vertebrates, are also present in the muscles of the fetus. The terms ‘‘red and white muscle” refer to macroscopical color differences only. Applied to the microscopic appearance of muscle fibers these terms become a misnomer if used as synony- mous with ‘‘dark and light muscle fibers. ”’ The interstitial granules and fat droplets of muscle are some- what uniformly arranged in longitudinal and in transverse rows between the muscle columns. Small granules and fat droplets form transverse rows in, segment J on either side of the membrane of Krause while those of larger size are in segment Q. The GRANULES AND FAT OF STRIATED MUSCLE 43 arrangement of the granules and fat droplets within muscle fibers is not dependent upon any connection with a fibrous net-work but is determined solely by the position of the membrane of Krause, the size of the granules, and the pressure of the muscle columns. The true interstitial granules are of a soft, plastic substance and have no limiting membrane. The exact chemical nature of the true interstitial granule is unknown. They are certainly not composed wholly of fat, though _ they contain an alcohol-soluble element. As was suggested by Regaud, they may be an albumino-lipoid. Many fat droplets in muscle fibers are not preserved by for- malin fixation. After a variable length of time in formalin fixa- tives the droplets may disappear. Fresh tissues must be used if all the droplets are to be demonstrated. This confirms a conclu- sion drawn by Bell. Fat droplets in muscle fibers are frequently stained but faintly or left colorless by the commonly used solutions of Scharlach R and Sudan rt in 70 per cent alcohol. Alkaline-aleoholic solu- tions of Scharlach R applied to fresh tissue stain all the fat drop- lets of muscle fibers. By this method preparations may sometimes be shown to be loaded with fat droplets when none are stained by the simple alcoholic solutions. Nile blue sulphate and Nile blue chlorhydrate color all the fat droplets of muscle fibers when fresh tissue is used and stained sections are placed in alkaline water or mounted in an alkaline medium. The droplets are usually colored red, but under certain conditions they stain blue. With favorable material, pectoral muscles of pigeon and bat, after a short formalin fixation, both true interstitial granules and fat droplets may be stained in the same preparation, the former blue, the latter red. Many fat droplets of muscle fibers are not blackened by osmium tetroxide. . For the most part the fat droplets of muscle fibers are neutral fat, glycerin esters of fatty acids. No convincing evidence has yet been presented to show that the fat droplets contain any substance other than neutral fat. They may not be pure neutral fat but it is improbable that they contain any considerable amount of albumin or other non-fatty substance. \ 44 H. HAYS BULLARD Both true interstitial granules and fat droplets are of wide dis- tribution in striated muscle, occurring under normal physiological conditions both in insect ,muscle, and also in the skeletal and cardiac muscle of vertebrates. The physiological significance of the true interstitial granules is uncertain. The quantity of fat in muscle fibers is increased when the animal (rat) is fed on fat meat and decreased during inanition. The fat droplets of muscle fibers are reserved food material. This conclusion was reached by Bell. This study was conducted under the direction of Prof. Irving Hardesty and I am under obligation to him for many helpful suggestions. I am also indebted to Prof. Gustav Mann who has given me valuable assistance. LITERATURE CITED AusBrecHT, E. 1902 Neue Beitriige zur Pathologie der Zelle, Deutsche path. Gesellschaft, Bd. 5. 1903a Uber triibe Schwellung und Fettdegeneration. Deutsche path. Gesellschaft, Bd. 6. 1903b Uber die Bedeutung myelinogener Substanzen im Zelleben. Deutsche path. Gesellschaft, Bd. 6. ALTMANN, R. 1894 Die Elementarorganismen. Arnot, J. 1900 Uber vitale Granulafirbung in den Knorpelzellen, Muskel- fasern und Ganglienzellen. Arch. f. mikr. Anat., Bd. 55. 1909a Zur Morphologie des Muskelglycogens und Zur Struktur der quergestreiften Muskelfaser. Arch. f. mikr. Anat., Bd. 73. 1909b Zur Morphologie des Glykogens des Herzmuskels nebst Bemer- kungen iiber dessen Struktur. Arch. f. mikr. Anat., Bd. 73. Ascuorr, L. 1909 Zur Morphologie der lipoiden Substanzen. Zieglers Beitriage, Bd. 47. Briu, E. T. 1909 On the occurrence of fat in the epithelium, cartilage, and muscle fibers of the ox. Am. Jour. Anat., vol. 9. 1910 The staining of fats in epithelium and muscle fibers. Anat. Rece., vol. 4. 1911 The interstitial granules of striated muscle and their relation to nutrition. Internat. Monatschrift f. Anat. u. Phys., Bd. 28. Benpa, C. 1900 Eine makro-und mikrochemische Reaktion der Fettgewebs- nekrose. Virchows Arch., Bd. 161. Ersensera, P. 1910 Uber Fettfirbung. Farbchemische und _histologisch- technische Untersuchungen. Virchows Arch., Bd., 199. Ewaxtp, A. 1910 Helle und triibe Muskelfasern beim Menschen. Miinch. Mediz. Wochenschrift, Jahrg. 57, No. 7. GRANULES AND FAT OF STRIATED MUSCLE 45 Faurt-Fremiet, M. E., Mayer, A., et Scoarrrer, G. 1910 Sur la microchimie des corps gras; application a |’étude des mitochondries, Arch. d’ Anat. mikrose., tome 12. Fiscuier, F. 1904 Uber die Unterscheidung von Neutral-fetten, Fettsiiuern und Seifen im Gewebe. Zentralb. f. allg. Path. u. path. Anat., Bd. 15 S. 913. Gritzner, P. 1884 Zur Anatomie und Physiologie der quergestreiften Muskeln. Recueil Zoolog. suisse, tome 1. Hemenuarn, M. 1901 Uber die Struktur des menschlichen Herzmuskels. Anat. Anz., Bd. 20. Herxueimer, G. 1901 Uber Fettfarbstoffe. Deutsche med. Wochenschrift, S. 607. 1904 Uber Fett-Infiltration und Degeneration. Lubarsch-Ostertag: Ergebnisse der allg. Path und path. Anatomie, Bd. 8. HoimaGren, E. 1907 Uber die Sarcoplasmakérner quergestreiften Muskelfasern. Anat. Anz., Bd. 31. 1907 Uber die Trophospongien der quergestreiften Muskelfasern. Arch. f. mikr. Anat., Bd. 71. 1910 Untersuchungen iiber die morphologisch nachweisbaren stoff- lichen Umsetzungen der quergestreiften Muskelfasern. Arch. f. mikr. Anat., Bd. 75. Keinaty, K. T. 1904 Uber den mikroskopischen Nachweis von Fett in nor- malen Muskeln. Inaug.-Dissert., Freiburg. Kemp, G. T. anp Hatu, L. B. 1907 The formation of fat in animals fattened for slaughter. Amer. Jour. Physiol., vol. 18 (Proceedings of the phys- iol. society). Kinessury, B. F. 1912 Cytoplasmic fixation. Anat. Rec., vol. 6. Kuorz, 0. 1906 Studies upon calcarious degeneration. Jour. Exp. Med., vol. 8. 1909 On the large white or soapy kidney. Jour. Med. Research, vol. 20. Knocus, V. 1909 Uber die Struktur der sogenannten ‘interstitiellen Korner’ (Kolliker) der Flugelmuskelfaser der Insekten, Anat. Anz., Bd. 34. Knouz, P. 1880-81 Uber Myokarditis und die tibrigen Folgen der Vagussektion bei Tauben. Zeitschr. f. Heilkunde., Bd. 1. 1889 Uber helle und triibe, weisse und rothe queregestreifte Muskula- tur. Sitz. d. Kais. Akad. d. Wiss. Wien, Bd. 98. 1891 Uber protoplasmaarme und protoplasmareiche Muskulatur, Denkschr. d. Kais. Akad. d. Wiss. Wien, Bd. 58. Knott, P., unp Haver, A. 1892 Uber das Verhalten der protoplasmaarmen und protoplasmareichen, quergestreiften Muskelfasern unter patholo- gischen Verhaltnissen. Sitzungsber. der kaiserl. Akad. der Wissensch., mathem.-naturw. Cl., 101. 3. Abt. K6éuiixer, A. 1857 Einige Bemerkungen itiber die Endigungen der Hautnerven — und den Bau der Muskeln. Zeitschr. f. wiss. Zool. Bd. 8; (cited from Retzius and Bell). ~ 1888a Zur Kenntnis der quergestreiften Muskelfasern. Zeitschr. f. wiss. Zool., Bd. 47; (cited from Retzius and Bell). 1888b Ueber den Bauder quergestreiften Muskelfasern, Sitz. d.Wiirzb. phys.-med. Gesellschaft. 46 H. HAYS BULLARD Koéuurker, A. 1889 Gewebelehre. 6. Aufl. 1. Kravusr, W. 1864 Die Anatomie des Kaninchens. Leipsig; (cited from Schaeffer). 1873 Die contraction der Muskelfaser. Pfliigers Archiv. f. Physio- logie, Bd. 7. Krausr, Rup. 1911 Kursus der Normalen Histologie. Berlin and Wien. Letivére, AvuG., tT ReTTERER, Ep. 1909 Des differences de structure des muscle rouges et blancs du lapin. Comptes Rendus Soe. Biologie, tome 66, f. 1075. McCrag, J., AnD Kuorz, O. 1910 The distribution of fat in the liver. Jour. exp. med., vol. 12., no. 6. Prenant, A. 1911 Problemes cytologiques generaux souleves par l’etude des cellules musculaires. III et IV, Jour. Anat. et Physiol. tome 47, no. 6. Recaup, Cu. 1909 Sur les mitochondries des fibres musculaires du coeur.C. R. Acad. Sciences, tome 149. Reaaup, C., et Favre, M. 1909 Granulations interstitielles et mitochondries des fibres musculaires striées. C. R. Acad. Sciences, tome 148. Rerzivus, G., 1890 Muskelfibrille und Sarkoplasma. Biologische Untersuch- ungen. Stockholm. N. F. 1. Ricker, G. UND ELLENBECK, J. 1899 Betriige zur Kenntnis der Verinderungen des Muskels nach der Durchschneidung seines Nerven. Virchows Archiv, Bd. 158. ScHarrer, J. 1893 Beitrige zur Histologie und Histogenese der quergestreiften Muskelfasern des Menschen und einiger Wirbeltiere. Sitz.d. Akad. d. Wiss. Wien, Bd. 102. Smitu, J. Lorratn 1906 The staining of fat with basic anilin dyes. Jour. Path. and Bacter., vol. 11, p. 415. 1907 On the simultaneous staining of neutral.fat and fatty acid by oxazine dyes. Jour. Path. and Bacter., vol. 12, p. 1. 1910 The staining of fat by Nile blue sulphate. Jour. Path. and Bac- ter., vol. 75, p. 53. Smitru, J. Lorrain, Marr, W. anp THorps, J. F. 1908 An investigation of the principles underlying Weigert’s method of staining medullated nerve. Jour. Path. and Bacter., vol. 18. p. 14. Smitu, J. LorRAIN, AND Marr, W. 1910 Further observations on the bichromate haematoxylin method of staining lipoids. Jour. Path. and Bacter., vol. 15, p. 179. 1911 Fats and lipoids in relation to methods of staining. Skandin. Arch. f. Physiol., Bd. 25. Srarkewitscu, P. 1894 Uber Veriinderungen des Muskel- und Driisengewebes sowie der Herzganglien beim Hungern. Archiv. f. experim. Path. u. Pharmakol., Bd. 33. Tuutrn, I. 1909 Morphologische Studien iiber die Frage nach Ernihrung der Muskelfasern. Skandin. Arch. f. Physiol., Bd. 22. VAN’ GeEHUCHTEN, A. 1889 Les noyaux des cellules musculaires striees de la grenouille adulte. Anat. Anzeiger, Bd. 4. Watspaum, QO. 1899 Untersuchungen iiber die quergestreifte Muskulatur mit besonderer Beriicksichtigung der Fettinfiltration. Virchows Archiv., Bd. 158. ON THE FATE OF THE JUGULAR LYMPH SACS AND THE DEVELOPMENT OF THE LYMPH CHAN- NELS IN THE NECK OF THE PIG ADMONT H. CLARK From the Anatomical Laboratory, Johns Hopkins University FOUR FIGURES In a study of the morphological changes which the jugular lymph sacs and the lymph channels in the neck of the embryo pig undergo during development, a number of questions must be considered. What are the primary lymph channels? Are they characteristic and constant in form? How are they modified during development? What is the correlation between the earliest lines of drainage and the drainage found in the adult? — What are some of the factors controlling these transformations? These and other questions arise. The purpose of the following paper is to make an analysis, and to offer a few epecesuions on the points mentioned above. The undertaking of this work was suggested by-Dr. Sabin, and it was through her kindness that this study was possible. There have been accumulating in the labgratory from previous studies a number of injections of lymphatics i in embryo pigs of all alaeen made by Dr. Sabin. These with numerous new injections have been cleared by the Spalteholz method,! and the present paper 1s based on a comparative analysis of these specimens. It has been the aim to give as accurately as possible the location of the lymphatics and the morphological changes in successive stages of development. There has been no attempt to describe the minute’ structure of the lymphatics but simply to trace the gross changes in the lymph channels. — 1 Spalteholz, W., Ueber das Durchsichtigmachen von menschlichen und tieri- schen Priparaten. Leipzig. Verlaz von 8S. Hirzel. 1911. 47 48 ADMONT H. CLARK The work of Sabin has shown that lymphatics first appear in the embryo pig 10 to 11 mm. long as an outbudding from the ante- rior cardinal veins opposite the third, fourth and fifth segmental branches. From these primitive buds a plexus of lymphatics is formed along the dorsolateral border of the anterior cardinal vein and this plexus is transformed into a non-muscular endothe- lial lined sac. From this primitive sae by continued centrifugal growth a large number of sprouts grow dorsalward into the pos terior triangle of the neck and form a complete arch of lymphatic capillaries connecting at either end with the primitive sac. This entire arch of capillaries becomes transformed into a part of the jugular lymph sae which explains the form of the final sac as shown in figure 1. From the jugular sac, still by centrifugal growth, the peripheral lymph vessels radiate forward over the head and backward over the anterior part of the body forming plexuses which are characteristic and definitely located For convenience I shall use the following terms in referring to the lymph sac, the form of which is shown in figure 1. (1) The anterior curvature of the lymph sac is the portion lying behind the pharnyx against the internal jugular vein. (2) The sac stalk is the portion of the sae also on the internal jugular vein extending between the point where the valve develops at the junction of the internal and external jugular veins and the ante- rior curvature. This is the first part of the sac to develop. (8) The apex is the portion of the sac lying in the posterior triangle of the neck. The reasons for this division of the sac are not obvi- ous in figure 1, but I shall show that they correspond to the func- tion of the three different parts of the sac. The apex of the sac connects with the sac stalk both through the anterior curvature and more directly by a large channel which joins the stalk not far from the valve into the vein. The form of the jugular sac is well shown in figure 1, which is a ‘diagram made from an embryo pig 2.8 em. long and in figure 2 which is a drawing of an injection of the lymphatics in a pig 3.5 em. long. From the sac four groups of lymphatic vessels develop. 2 A part of this work is in the Amer. Jour. Anat., vol. 1, 1901-1902, and a part of it will be published in the Ergebnisse fiir Anat. und Entwicklungsgeschichte. JUGULAR LYMPH SACS AND CHANNELS—NECK OF PIG 49 The first group consists of a few large vessels which have grown from the apex, the most superficial part of the sac, over the scapu- lar region. The second place of growth is the dorsal border of come any ii AG ES =: aS SS. SS2 CC. Fig. 1 Diagram of the jugular lymph sac in an embryo pig 2.8 em. long to show the points of origin of the peripheral vessels. X 10. A, apex; A.C, anterior cur- vature; C.C, cross connection between the apex of the sac and the sac stalk; C.P. superficial cervical plexus; O, occipital lymph duct; Pa.T.F, point of origin of the posterior-auricular, temporal and facial lymphatics; Rp, retropharyngeal lym- phatics; S, stalk of the see; Sm, submaxillary lymphatics; S.S, primary supra- scapular lymphatics; S.S2, suprascapular lymphatics from the cervical plexus; T.B.L, thoracie and branchial lymphatics. the apex just anterior to the suprascapular vessels. A large duct extends forward over the occipital region of the head. ‘This particular vessel is very large in the human embryo as can be seen THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO 1 50 ADMONT H. CLARK in figures 505 and 506 of the Handbuch der Entwickelungsge- schichte des Menschen. Keibel and Mall., vol. 2, 1911, ps. 708-709, after Sabin. The third group of vessels is from the anterior cur- vature of the sac where it arches dorsalward and lateralward behind the pharynx. The importance of these vessels is shown both by their size and their early appearance. In an embryo 2.8 em. long the anterior curvature has a distinct bulge protruding toward the buccal cavity, and in one specimen a few ducts can be seen radiating toward the pharynx. From this retropharyn- ° geal process of the sac are to be developed all of the lymphatics of the pharynx, Eustachian tube, the nasal cavity and a part of those of the tongue. The fourth group of vessels is by far the largest. Infigure 2 will be seen a group of vessels from the ventral border of the apex of the sac which grow ventralward external to the sterno-cleido-mastoid muscle and form an extensive lym- phatic plexus along the course of the external jugular vein. This plexus I shall call the superficial cervical plexus since it gives rise to the superficial cervical lymph glands. The injection shown in figure 2 is not a complete injection for these vessels. The point of injection was in the suprascapular vessels which is an indirect point for the superficial cervical plexus. The vessels from the ventral border of the apex of the sac are present in an embryo 18 mm. long and hence they begin at about the same time as the suprascapular lymphatics. The superficial cervical plexus as shown in figure 2 has already sent a group of vessels cranialward, part of which are shown as posterior auricular lymphatics. The vessels which grow forward along the external jugular vein divide into two groups,’ the temporal and the facial. From the ventral border of the superficial cervical plexus develop Fig. 2 Injection of the jugular lymph sac in an embryo pig measuring 3.5 cm. long. Magnified about 10 times. This is the same specimen which was shown as figure 3 in The American Journal of Anatomy, p. 186, vol. 3, 1904. The specimen has since been cleared by the Spalteholz method so that it shows the relation of the superficial lymphaties to the jugular lymph sac. It is a complete injection of the suprascapular and occipital plexuses and an incomplete injection of the begin- ing cervical plexus. The sac stalk shows faintly where it extends internal to the arm. F.v, facial vein; a little blood in this vein enables one to locate the position of the superficial cervical lymphatic plexus. 51 NECK OF PIG JUGULAR LYMPH SACS AND CHANNELS RRORRSwE | 52 ADMONT H. CLARK as shown in figure 1 the lymphatics for the skin of the neck and the submaxillary vessels, while the caudal end of the plexus gives rise to the superficial lymphatics of the arm and of the thoracic wall. Thus the jugular lymph sac gives rise to the suprascapular, occipital and pharyngeal lymphatics directly and is the place of origin of the superficial cervical plexus which in turn supplies all the rest of the lymphatics for the head, face, neck, thorax and arm. The deep lymphatics of the arm have not yet been worked out in the pig. In the cat and in human embryos they arise from an extension of the jugular lymph sae which lies along the primi- tive ulnar vein. } These fundamental groups of lymphatics the suprascapular, occipital and superficial cervical, which can be seen in the embryo 3.5 em. long and indeed can be injected a short distance from the lymph sac much earlier namely in specimens measuring 18 mm. are constant. Ducts originate from definite places and establish definitely-localized plexuses. Thus in the early stages there are distinct plexuses in the skin which are connected with each other only through their central connection with the sac. Such a primary plexus for example is the occipital plexus of figure 2. By subsequent development however, these separate areas become interconnected, so that an injection instead of being lim- ited to one of the primary plexuses spreads out quite widely, reach- ing the sac not by a single set of ducts but by a number accord- ing to the extent of the injection. Thus, plainly, the earliest lymphatics drain definite areas which are distinctly located and definitely defined. In figure 2 it is shown that the suprascapular vessels drain by a few vessels sometimes not more than one or two directly into the apex of the sac. At this stage there are a few small anasto- moses between the suprascapular vessels and the superficial cervical plexus. These anastomoses are destined to become very abundant so that there are eventually more vessels which connect the suprascapular plexus with the superficial cervical plexus than with the primary sac. This process of the development of anastomoses between the different primary plexuses goes on until the entire superficial JUGULAR LYMPH SACS AND CHANNELS—NECK OF PIG 53 lymphatic plexus is a complete layer of lymphatics covering the body. This stage is shown in Sabin’s figure 5 in The American Journal of Anatomy, p. 188, volume 3, 1904 In this figure it is not possible to analyze the primary plexuses; the suprascapular, occipital, posterior auricular, temporal, facial, cervical, thoracic and brachial vessels make one continuous plexus. When an injected specimen of this stage is cleared however by the Spalte- holz method the place of origin for each plexus can be made out. In figure 3 it is clear that the primary lymph sac has the three divisions already given, namely, the apex in the posterior triangle the anterior curvature and the sae stalk lying deeper and hence showing very faintly on the internal jugular vein. The apex of the sac and the anterior curvature may now be called lymph glands, the sac stalk however remains as the deep jugular lymph trunks. In comparing figures 2 and 3 it is clear that in the earlier stage the outline of the sac is a comparatively smooth curve from the stalk around to the apex. In an embryo pig 20 mm. long the entire dorsal border of the sac has a series of sprouts but the permanent ducts are however limited to certain areas along the sac and these parts enlarge while the intermediate parts remain small. This determines the position of the lymph nodes. Three factors seem to guide this primary node formation in the sac. First the apex of the sac receiving as it does the suprascapu- lar and occipital vessels directly and all of the vessels of the face, neck, arm and thorax indirectly through the superficial cervical plexus is the largest center of drainage in the neck. Lymph glands develop at the centers of drainage and the apex of the sac therefore becomes an early and alarge node. Second the portion of the sac between the apex and the anterior curvature can be assumed to be comparatively non-functional as a path for lymph conduction, for the apical drainage would most easily pass to the veins by way of the cross connection to the stalk. Hence the por- tion of the sac intervening between the apex and the anterior curvature remains small. Third, the development of the sterno- eleido-mastoid muscle which crosses the sac between the apex and the anterior curvature probably causes a pressure to be exerted at this point. Drainage, function and structural relations can 54 ADMONT H. CLARK be said to be factors in controlling node formation and develop- ment both in the lymph sae and along the peripheral lymphatics. The relations of the peripheral lymphatics to the sac and the development of nodes thus described are constant. The superficial cervical plexus needs a very careful description. It is clear that it is an important structure since it drains so large lig. 3 Injected jugular lymph sac, superficial cervical lymph plexus and the peripheral lymphatics in the neck of an embryo pig measuring 5.5 cm. long. Mag- nified about 7.5 times. This figure is to be compared with figure 5in The American Journal of Anatomy, p. 188, vol. 3, 1904, which is a complete injection of the super- ficial lymphatics of the same stage. A.s, the apex of the jugular sac making the lymph gland of the posterior triangle; A.c, anterior curvature of the lymph sac making the deep jugular lymph gland; C.p, superficial cervical lymph plexus; S.g, submaxillary lymph gland; S.s, stalk of the jugular lymph sac. JUGULAR LYMPH SACS AND CHANNELS—NECK OF PIG 545) an area. As seen in figures 3 and 4 it is an extensive and dense plexus of lymphatics lying along the external jugular vein lateral to the sterno-cleido-mastoid muscle. It has in reality two points of origin. First, the ventral border of the apex of the sac shown in figure 2 for an earlier stage but still better in figure 3. The second place of origin is a plexus of lymphatics from the stalk of Fig. 4 Injected jugular lymph sac and cervical lymph plexus in a pig measur- ing 7.5 cm. long, to show the relation of the developing glands in the neck to the sac. Magnified 6.5 times. A.s, apex of the sac or gland of the posterior triangle of the neck. The anterior curvature of the sac, which is a deep jugular pharyngeal lymph gland, shows behind the sterno-cleido-mastoid muscle. C.p, superficial cervical plexus which is destined to be a group of lymph glands. At the cerebral end of the plexus is a developing facial gland. S.g, submaxillary lymph gland. 56 ADMONT H. CLARK the sae which follow the external jugular vein. This group of vessels is indicated by one trunk in figure 1, but the vessels show much better in a mesial view. Mesial sections of pigs 5 to 6 em. long show that there is an abundant plexus of vessels arising from the stalk of the sac in the root of the neck near the place where the lymphatic sac connects with the vein. < eee eee Gray fox (Urocyon cinero-argentatus)..... IbymersCPelis TULUS) Qe. .<.a tie ss os. 9 ee Raccoon (Procyon loctor)...........seeoee Rhesus monkey (Macacus rhesus)......... Opossum (Didelphys Virginiana)........... Cat/(Melisidomestica):.. 0 ....... 08 Spider monkey (Ateles paniscus).......... Java monkey (Semnopitheous maurus).... Rabbit (Lepus cuniculus)....:......2eesee Agouti (Dascyprocta agouti).............. Guineéa-pig (Cavia copaya)................ Gray rat (Mus Norvegicus)............... White mouse (Mus musculus, albus)....... Bat (Nyctinomus brasiliensis nasutus)... 3,628.736 566.990 430.910 136.077 68.038 72.574 34.019 8000 1250 950 300 150 160 75 PRINCIPAL REGIONS OF SPINAL CORD AD TABLE 2 Measurements of cervical region in millimeters. Table 2 includes measurements of transverse sections from the cervical enlargement. The numbers in the heads of the columns correspond to the lines of measurement so numbered in text figure A, and the figures record actual size of the sections. ~ | : | 2 aR | E 4 ‘ ANIMAL eres eaeeeilmm (Se | | ws le | ag 4a 3 ao | Pe ae a ara ES Elephant........... _..|17.06 |29.78 |23.42 | 8.21 |13.68 |10.94 /6.00 | 4.43 | 1.12 Horse................|13.48 |23.28 |18.35 | 7.28 | 8.81 | 8.04 | 4.00 | 2.50 | 0.68 Ox 11.15 |17.85 |14.50 | 5.56 | 7.18 | 6.37 | 3.84 | 2.60 | 0.56 Bear it 0) BEB) UAB) |) Doe |) 7/7 Oats || Sei I WACO) Woe [SIGs © Recent ener 9.71 |11.00 |10.35 | 5.18 | 4.00 | 4.59 | 2.62 | 1.18 | 0.43 NICHOL, © Baca et eet pe ee 8.93) 13981 111137) 4 osae mel |, 568) (2275) 1.87 0162 Sheep................| 8.92 {11.18 |10.05 | 4.87 | 5.12 | 4.99 | 2.78 | 1.85 | 0.75 Kangaroo..... 502 8. 84> SP, | 8258 onl? || sash) |) 12437) 1a 75 O87 Ape... GLOOM ete Lae slon| ShO2monOze| ana) | le S2 ele Zn tORGZ Dog.. Noelle Grol | vo nOOmmeesialraeOo) | 20m 2e Orsi Roxane. Vl .fe | C18 | 7.57 | 3:87 3-46 | 3.667). 1-81 | 1.03.1°0.62 yah Pa ete bathe Ae 9.43 |10.71 |10.07 | 4.98 | 5.87 | 5.42 | 2.62 | 1.60 | 0.50 RACCOON Es ae aan: Hoa N Cok |) CG Wi 4eeal | 3.055 |) BO) Wea as) I) O88 Rhesus monkey......} 4.81 | 6.50 | 5.65 | 3.00 | 2.93 | 2.96 | 1.75 | 1.25 | 0.56 POSSI: freee. - AN62))/16. 115) |, 5. 38: 2602) |eon06)| 2.84.) 1439) 1221 | Or3F COMhasnecaconsoseoasedl OOO) | VU. 2834.43 |! sai Beto raya) teal es) Spider monkey....... 5.68 | 7.00 | 6.34 | 3.70 | 4.12 | 3:91.| 1.84 | 1.59 | 0.62 Java monkey......... DLOSeOwOle | Oe 99 aeons om lace OOM ler Oli mle OG fates 12 TRIO. cncobeee co eee | CaCO Dea IT ae) | ee) |) Zt 10) Staats) ] alae) | SOM ae Aine oe eno eel Gard), 6.43.|-6.57 | seogmuaecon! oe42 (b50)) 1105) 0.37 Guinea-pig...........| 2.87 | 4.06 | 3.46 | 1.93 | 2.18 | 2.05 | 1.12 | 0.81 | 0.48 ya eee ee ORONO N TAO roe On MO Miners salons litt) OKs ti. On4s Be cstise ss Se anaes 8) 1.48 | 2.56 | 2.02 | 1.09 | 1.68 | 1.38 | 0.78 | 0.75 | 0.25 LEACH Horas Creat aN eee eae ee SI 29S ez 2a lem OSs leo 9) OL93 0.87 | 0.15 | | | 78 PEARL BRIGGS BULLARD TABLE 3 Measurements of thoracic region in millimeters. Table 3 records measurements of transverse sections from the thoracic region, taken as those recorded in table 2. — j — ———— —__—____— = 3 8 2 2 re 5 2 ba ANIMAL $8 | ag 5 ae Be ze By Ey ee Ba | 38 | Bo) 8s | be | Be | ee | ee | ee Ble phanhr vcs sss 14.65 |18.56 |16.60 | 5.71 | 4.68 | 5.19 | 1.10 | .1°75 "0075 THOISC. pened eee) os 2h 20 13s 25 9) E22 7S por OUR oma Onell lon lela mOnGe Ox oo. be Sone. esl 987 [11.18 110.525) 2Se855) cs00M 242 9) 1-01) ae eas Bear.. 6:87'| 8.12 | 7.49 | 22 3R 2812 56))0- 84.) 0.87 oie ts VG Sige cree te. ee Anca ts 7.00'| 7.56-| 7228) | 32125) S68n 2240) 02627) 0. odes MVipinace eed ee ce, oe ahs 7.31 | 8.50 | 7.90 | 2.68 | 2.43 | 2.55 | 0.93 | 0.50 | 0.37 DHCCD a see al Oran 6:62 | 6.46 | 3.12 | 1.87 | 2.49 | 0.56 | 0.62 | 1.06 Kangaroo..... 6.81 | 7.15°| 6.98") 2387) 1.56") 2/21-! 0.50 | 0.56.) dase Ape... 4.15 | 4.68 | 4.41 | 1.75) 1.18) 1.46 |-0.32 | 0.87 | 0:56 WOR Sa tweens eee. 3.76 | 4.59 | 4.17 | 1.62 | 2.09 | 1.85 | 0.65 | 0.53 | 0.56 Fox.. 5.15 |) 5.067 |b: LOM S2eSe leon lal Wi OL465 fOR2bR tOReS ymeut Sica Oem... 5.35 | 6.50 | 5:92 22712 | 187 |) 1°99 | 0:64 | Ostia aie ACCOODEEER ee eee 4.62 | 4.87 | 4.74 | 2.03 | 1.15 | 1.59 | 0.40 | 0.53 | 1.00 Rhesus monkey......| 3.37 | 4.93 | 4.15 | 1.387 | 1.48 | 1.40 | 0.40 | 0.62 | 0.93 Opossum: =. fo: 60 5 ats. | 3.60 | 3.93 | 3.76 | 1.75 | 1.18 | 1.46 | 0.16 | 0.43 | 0.56 Cat. cccet. soeee sss eel 4:18 | 4.86 | 4252S tevos £76: |.0.. 505 | (0) G2 aime Spider monkey....... 4512 | 4.12) 4.122 St tots 740.28 | 0y43 see Java monkey......... 3.01 | Sc 71 ese ele 9GR ON GSe noe | Oko ves Oe2on mieten Rabbit.....0........0.| 8.84 | 4.53) 4.18) 2 Oe 1 162) Lait ) 064 | OC 564.0 rre Agouti...............| 5.12 | 5.37 | 5.24 2:31 ) 1.189.100 74 | 0.50 | 0.56 | 0:48 Guinea-pig...........| 2.78 | 2.87 | 2.82) 1.78 | 4.12 | 145 | 0:45°| 0.65 |Orem RAG. cic cs esses cel 2.81 | 2.62. |°2 Cie rom uaa alesse Oe.) OSisa ate Mouse...............] 1.31 | 1.56 | 1.43] 0.84 | 0.68 | 0.76 | 0.28 | 0.43 | 0.37 BU tite eteal de taxi me al he ge 1.59 | 1.43 | 1.00 | 1.25 Pea OL Sl One Obes PRINCIPAL REGIONS OF SPINAL CORD TABLE 4 Measurements of lumbar region in millimeters. transverse sections from the lumbar region, taken as those recorded in table 2. 79 Table 4 records measurements of Hee | |’ | 3 | = | I es ‘ | z P| (2 5 nf a z | & z Ba [here | sale s | ae | 3 Bee lee 8 5 ANIMAL (21 a= ra) ze Q Oy n ma.) g&8 Ze Be Ze | Bz By BS Seve ce lh Sonesta | So] Be | gel ee Aeon ee eetee | Be | Fa.| Fe | eS Horse eeals tage Te Ge Also te ONS | 9.21 | 5.31. |3.70 | 0.50 Ox. at) 9.18 |16.00 |12.59 | 5.84 | 9.00 | 7.42 .)3.75 | 3.37 | 0.50 Bear ..| 8.93 |10.46 | 9.69 | 5.00 | 5.87 | 5.43 | 2.83 | 1.65 | 0.75 [BIOTA Ge aD OB eC | 7.56 | 9.56 | 8.56 | 3.68 | 4.75 | 4.21 | 1.62 | 1.40 | 0.68 1 Se | 8.53 | 9.50 | 9 Ole 49st eaes7. | a05 \2.505) 1.81 | 0.62 BHCED en eee gel) 29) |10).31. |S 580") 4.3% |e. 50 | 4.93 | 2.71 | 2.10 | 0.62 Kangaroo...... .| 8.45 |10.12 | 9.28 | 4.65 | 4.25 | 4.45 | 2.65 | 2.00 | 0.56 Ape. | 4.92 | 6.28 | 5.60 | 3.00 | 4.25 | 3.67 | 1.90 | 1.43 | 0.37 WONG arse ok se es GPP | 4.75 | 6.34 | 5.54 | 3.21} 4.06 | 3.63 | 2.12:| 1.32 | 0.50 Fox eee eaionad |) f2 12 |.6.71 | SeOlaeaeSh 23.81 | 168)| I18: 0756 nese see NS es st: | 7.81 | 9.65 | 8.73 | 5.12 | 5.78 | 5.45 | 2.45 | 1.67 | 0 33 TAC COON 252) Rages ae | 6.21 | 6.81 | 6.51 | 3.71 | 4.50 | 4.10 | 2.09 | 1.43 | 0.71 Rhesus monkey...... 450, | 5.81 | 5.15)| 3:06-/'3.37 | 3-21 | 1.65 | 1.25 | 0.50 @yossume sce 4 2,2) 4.514| 5.56 | 15-03 | 2°93. 8am12, | 3.02.) 176 || 1.46 |0.,71 Ate ee ee 20-8 | ©. 43 | 5.87 |3- 43) eaan0 | 3.46 | 1.75 | 1.43 | 0.37 Spider monkey.......| 5.46 | 5.56 | 5.51 | 3.50 | 3.87 | 3.68 | 1.93 | 1.78 | 0.62 Javamonkey.........| 4.75 | 5.75 |°5.25 3.62 | 4.06 | 3.84 | 1.87 | 1.37 | 0.87 Rabbit...............] 4.02 | 6.14 | 5.08 | 2.65 | 4.12 | 3.38 | 1.89 | 1.25 | 0.56 PE POUtine ees atm.) 02908|. 6280 | 6.388 | Sodomen 200) 4.40.1 2.374 | 175: | 0.7) Guimea-pie-s-. ce. .2| 2-80-! 3.87 | 3.36") 1:96 2.37 | 2.16 | 1.16 | 1.15 | 0.31 Rains eee t| 2.09 | AL30 163.45 | DET 87 22329" | 11.81 | 0.87" |.0/35 RV GMISG co20hSe § ooo « 1-45 2712) 1.78) Oss Ie 37.) 1.20. )70..65: | 0.60 | 0:35 Ds tendon crass 3.5 Dols 2725 11.78 | Daee 1 56)) 12334) 0275 | 0.81 | 025 SO PEARL BRIGGS BULLARD OBSERVATIONS Common structures In glancing over the drawings, one of the first things which attracts attention is the marked similarity of form throughout the series. Each cord is possessed of bilateral symmetry and presents a grey figure in the shape of the letter H, at times con- siderably modified. Each half of the grey figure of the cord has the ventral and dorsal horn, the gelatinous substance of Ro- lando being found at the apex of the latter. The two sides of the grey figures are connected by a commissure in which is: a central canal surrounded by a homogeneous substance (central gelatinous substance) similar in structure to the gelatinous sub- stance of Rolando. The white substance surrounding the grey is divided into a dorsal and a ventro-lateral funiculus by the more or less well marked dorso-lateral sulcus, the line of ingrowth of the dorsal root. The dorso-intermediate sulcus pro- vides an interesting comparison which will be mentioned when the dorsal funiculus is considered. Each section shows a ventral median fissure but not all, in Weigert sections at least, presents a dorsal median septum. The reticular formation, present in all, varies considerably in amount as the drawings show. Diameters Text-books on neurology state that the human spinal cord in each region has a lateral diameter greater than the dorso-ventral. Authorities agree that the greatest difference in these diameters, or in other words, the portion of the cord most flattened dorso- ventrally, is the cervical region. Cunningham ’09) states that the lumbar region is more nearly circular than the thoracic. Piersol (10) on the other hand, gives the lumbar region as being more flattened than the thoracic. The measurements for man here given agree with those of Cunningham. However measure- ments taken from several other specimens in this laboratory agree more nearly with Piersol. From table 5, which records in the form of a ratio the relative amount of flattening in each region, it is seen that the bat has a PRINCIPAL REGIGNS OF SPINAL CORD 81 cord more flatttened in its cervical region than any other animal in the series here studied. This may be accounted for in part by the extensive innervation required by the greatly developed wing musculature. The thoracic region, in general for all the spinal cords, is flat- tened dorso-ventrally. In most cases the flattening is much less than in either the cervical or the lumbar region. The cords of the agouti, lynx, and rhesus monkey are peculiar in that they TABLE 5 Ratio of dorso-ventral to lateral diameter. Table 5 gives the ratios of the dorso-ven- tral to the lateral diameter obtained by dividing the figures in column 2, tables 2, 3, and 4 by those in column 1 of the same tables. Thats to say, the dorso-ventral diameter is to the lateral diameter as 1 is to the figures here recorded. In each column the animals are arranged in the order of the dorso-ventral flattening of the regions of their spinal cords, those showing the greater dorso-ventral flattening being placed first. The ratio for the lumbar region of the elephant is obtained from diameters given by Kopsch, as quoted from Hardesty. CERVICAL THORACIC : LUMBAR 1 ; 2 | 3 TEE eco beia ee Sa Ee 2 29)| Ehesus monkeysyeieee0. | OX os ee es S174 Piepoant...o....0...--le¢4 | Elephant... 02. HO (babes. Soke ee lend new mare Hear le Batenta..0 ss segeleeds | Rates: Se See Te 6G INIOUSE Re ior Beas L2H) Orns coh seere M225) SEIOTSE ..: sorasaucie ch oe 1.65 (D5 SERS Cie Be rice ee Cie RUS a a Bg 0. ape MN RAD DME: scree: 0-5. 408. LOS Gree ere aes, a) oD) | MLOUBE:.. x. 5.55. oon Mebos Tlepmant =. oo. 6s «2%: 1.52 1M Ts a en RON ee OE Wh GAELS corse haat EOE CVLOUBEE i: Ghton teins 1.46 1D (yee ce ee Mae iea9? tab bits. 2: <0) oaeletael SHEED Yc). cece: sls 1.41 Guinea-pigee ane.) ait Maney Go. et PalGriGuimes=pig 2.25.02 85 ebesismMonkeye tse seele oor! Cat icn...s 5 kee OM DOP). ok. ae lig a Re 1.33 CpossuMies meee elias) OXY... asine 5. ele. Rhesus, monkey....\..... 31.29 SEC Bese ne nen NADIE 3.3... 5 «2's eee ADE. Seale. LOT Spivecrmonkeyets. 5. ..25| OPOSSUM. .:. 5:8 welrOOn HOP. ; co.es ss hace. 1.26 Pe aie a Reet tee ora et Dos SEL OGs 0',, 3°.’ os SEA AOS pin Ki 8 SAP) eek ROT ARES. 1.23 ao lt een asa feos t 2k) EL OSG. « +.4. 244. ee eleOS | OPOSsUMN ene oa ace). be 23 Mitre ee ee ae We OPl Raccoon. 5. 4.8 oe Lite CBS"I IN GE Agno ce ess eee | Kiauearog 7.4... 2d 7. || Kangaroo....,5 5.104 | Java monkey... ....,.. 1.21 Ape. Ror aeea al LG VAPOUR as ce eel Os Kanoaroore:......+<.. 2:19 LUN AaB: Cceesas eo caren CED ASA SHCEDiat At vee eREOs Ih Beaty 2282 0 Hh cae tal RT BIG (a) Dee a eee 1.13.) Guinea-pig........ ARO WAG OUT ec ct) ys lal 6 Javal monkey .5....... fell Spider:monkeyseeu OOo hi BOX. occas deem sivas cde Ox eer heck sc 1.05 | Java monkey..... THOOR EWMiamiie er sec ee isa UAECOGI mete cib acres « WADA Oe ot.) 9 Shieoe ee ORGS a RACCOOM: sehh |). osc am 1.09 AP OUTIR aan ote’ 0:95) | Ratije.s.as. +) <. 0090) "Spider monkey. :..):.... 1.01 82 PEARL BRIGGS BULLARD possess greater flattening in the thoracic than in the cervical region, the sections of which latter are approximately circular. Man, bear, and rhesus monkey stand alone in having a cord in which the thoracic region possesses more dorso-ventral flattening than does the lumbar region. In about half the mammals of the series, including man, the cervical region of the cords shows a greater dorso-ventral flat- tening than the lumbar region. In the other half the order is reversed, the greater flattening occurring not in the cervical but in the lumbar region. Chief among this latter number are the sheep, rabbit, hog and Java monkey which, as will be noted, have relatively a very small dorsal funiculus. While on the other hand the bat, mouse, and man have greatly flattened cervical regions and very large dorsal funiculi (column 4, table 8b). The shape of the cords in the series varies to such an extent in each region that it is difficult to say that any given cord is of a pre- dominately rounded or flattened form throughout its length. However, from data here given we may say the bat and elephant are types of dorso-ventrally flattened cords while the fox pos- sesses a cord of rounded form. Enlargements and total area . As is well known the cervical and lumbar enlargements are the result of a response to the increased demand for innervation made by the extremities. Furthermore, four-footed animals with approximately equally developed extremities have the area of the cervical enlargement greater than that of the lumbar, due largely to the fact that the cervical region is concerned with the innervation of the tissues of the thorax in addition to those of the upper extremity as well as to the fact that this region carries all the fibers connecting the regions below it with the brain. The kangaroo, with its very small anterior extremities, has a lumbar enlargement the total area of which in transverse section is 13.99 sq. mm. greater than the total area of the section of its cervical enlargement (column 1, table 6, and fig. 8, C and i); PRINCIPAL REGIONS OF SPINAL CORD 83 Schmidt (08) states that the Dipus (laculus), a kangaroo- like rodent, has, notwithstanding its relatively small anterior extremities, a cervical enlargement which exceeds the lumbar. He suggests that the size of the cervical enlargement may be due to the relatively more active anterior extremity. A very striking illustration of the significance of the enlarge- ments is given by Streeter (’03) in a description of the ostrich cord. The ostrich, according to Streeter, has an insignificant cervical enlargement to correspond with the almost functionless wings. The ‘lumbar brain,’ on the other hand, extends through eleven segments and its transverse section has an average area of 38.5 sq. mm., which is 20.3 sq. mm. greater than the largest area from the cervical enlargement. Hardesty (05) finds a similar condition in the cord of the emu, a bird closely allied to the ostrich. The ostrich and emu may be taken as examples in. the bird family, of a condition present in the kangaroo among mamumalia. Spitzka (86) states that the bat has an insignificant lumbar enlargement to correspond with the diminutive posterior extrem- ities. The specimen of bat here used (Nyctinomus brasiliensis nasutus) has a well marked enlargement in the lumbar region as well as a large cervical enlargement which furnishes inner- vation for its powerful wings (figs 24, C and LZ). Likewise, it is interesting to note, as stated by Cunningham (’09), that in the cetacea, although there are no visible hind limbs there is a well marked lumbar enlargement. In these animals, this enlarge- ment is no doubt required by the large and powerful tail. The grey substance The H shape of the grey figure, described as characteristic of the human cord, holds, as is well known, for the great major- ity of mammals. In the thoracic region of the bear, rhesus monkey, cat, dog, lynx, and spider monkey the H is highly modi- fied (Th in figs. 4, 10, 12, 14, 16, 17). In the first five animals the peculiar shape consists in a shortening or flattening of the dorsal horns and a relatively very wide grey commissure. The almost complete absence of the dorsal horns in these animals 84 TABLE 6 Areas of transverse sections in square millimeters. in square millimeters of the transverse sections from the different regions of the spinal cords of the animals given and actual areas of the portions of the transverse sections as indicated in the headings of the columns. | | | | | | 9 AREA OF ANIMAL REGION | eer | _onar Fae. 5 429.68 | 74.06 Hlephantiseetn ov aed. | T: 224.06 | 16.40 Ir, C. | 262.96 | 44.53 Horse). eos oe | TN. 131.25 14.06 L. BS} BD) 62.50 (Ce 167.815 |) 336287 Race te tye Me ss Ts 85.93 | 9.37 i. 132.038 39.53 Cr 120.15 29 .92 IEICE Dass eee eee eieiee nica TT. 44.03 4.92 L. 74.60 21.56 @: 85.00 18.59 PLO ck teak ete et: ADE 36.40 4.14 ila. 59.92 TES Cc: 100.39 16.75 Man als 52.42 5.00 1B 65.93 Pal Ae C. 82.65 PALATEAL Sheepreec ete es ‘40F 35.70 4.37 L. 61.70 20.15 (Oy 55.93 10.62 Kangaroo ale 40.46 3.75 1b. 69.92 19.68 C. 41.64 10.23 INDO See seed Shy oe aT 16.25 1.56 L. 26.09 10.54 Ci 35.46 10.93 1B foe dro entiyt MARY eet ae oe ae “he 14,21 2.65 L. 26.25 9.68 (OF, 45.15 11.79 Lay cote Ree Als 21.40 2.26 L. 37.18 At S7Al cs 80.37 22.03 Lagias freee. shee AG 27.89 3.20 Li: 53.90 | De 25 PEARL BRIGGS BULLARD 4 Table 6 records the total areas | 3 | | 5 | ‘Sout _| ANTEX” ("op amr | FUNICULUS PUNICULUS | SUBSTANCE 1100.00 | 255.62 | 355.62 40.938 166.73 207 .66 47 .34 171.09 218.48 | 15.15 102.04 117.19 | 51.56 119.29 170.85 | 29.06 101.88 130.94 9.68 66.88 76.56 21.87 70.63 92.50 20.70 69.53 90.23 ion 31.54 39.11 16.64 36.40 53.04 11.59 54.82 66.41 4.92 27.384 32.26 13.438 35.24 48 .67 26.17 57.47 83.64 14.53 32.89 47 .42 15.62 29.18 44.81 11.56 49.38 60.94 3.12 28.21 3lieo 9.21 32.35 41.56 9.60 35.71 45.31 4.21 32.50 |. 36.71 12.42 37.82 50.24 8.82 22.59 31.41 ae IAS 14.69 4.60 10.95 15.55 5.78 18.75 24.538 1.56 10.00 11.56 3.90 12.67 16.57 6.25 Dil 33.36 2.08 ivéaitil 19.14 4.68 20.79 25.47 14.06 44.30 58.36 3.20 21.49 24.69 9.29 23.36 32.65 PRINCIPAL REGIONS OF SPINAL CORD ANIMAL REGION TABLE 6—Continued | RACCOON ste Rhesus monkey...... Opossum 6... - (CEASE OB ee Ae eae Spider monkey....... | Java monkey........ | ReaD bits. a. set raiies-pig in. 3 Fk... GRAY EA ef SS. ME OP HOPHOPH OPH OPH OPH OPH OPH OHS ORHNOEE Ne) NWO De Noe wow . 20 | AREA OF | 9 GREY | FIGURE js jm “I 0 “I — Ne fer) Or — — pt _— BrPNrFOrFWAOrE PRE WONAWANNAHEKHOONNONODORAAH SO On (=) — — 3 FUNICULUS — OOooeoeoOFP KP KE KP KH PWRMANHEH NNER WORNONHE BRH OO WOd 70 82 39 62 o4 37 68 56 50 70 93 4 AREA OF AREA OF | DORSAL ANTERO- | LATERAL | FUNICULUS | bo bo me Re HO bdo bo “Ie sI e © N ST = ~I 0 — “Tho © © bo C1 wane OS — ~w) b ceili ancl xiii andl on’ cor NW OO © (o/) i) 15.63 85 5 TOTAL AREA OF WHITE SUBSTANCE 33.52 15.94 PAL Ai 17.89 11.25 13.44 17.50 86 PEARL BRIGGS BULLARD is quite striking. This condition may be interpreted as a dorso- ventral thickening and mesial fusion of the dorsal horns (posterior grey columns) since the gelatinous substance of Rolando and the dorsal horn cells are present as normally. The spider mon- key (fig. 17) possesses relatively short ventral or anterior horns and this relative shortening is evident through all three regions of the cord. ' In all the animals here studied, with one exception, the width or thickness of the ventral horn in the cervical region exceeds that of the dorsal horn. This exception is in the kangaroo in the cervical region of which, the caput of the dorsal horn is wider than is the ventral horn. This condition in the kangaroo is due, not to a relatively extra wide dorsal horn but to a narrow ver- tral horn. Because of its small anterior extremities, 1t isproba- ble that cutaneous innervation may not have decreased to the same extent as the innervation required by the muscles, which receive motor or ventral root-fibers and which have atrophied through greatly lessened use. If such be the case, the retained width of the dorsal horn may be understood in that it contains the cell bodies of association and commissural neurones about which the dorsal root or sensory fibers terminate for purposes of functionally associating different levels and the two sides of the spinal cord with sensations brought into the cervical region. The lumbar region shows throughout the series the ventral horn to be wider than the dorsal horn (columns 7 and 8, table 4). However, the average difference in the width of the two horns in the lumbar region for the series is only 0.52 mm. while in the cervical region the average difference is 0.64 mm., the ventral horn being the wider. The difference in width of the dorsal and ventral horns is in the thoracic region very much less than in either of the other two regions (columns 7 and 8, table,3). In most cases the dorsal horn is somewhat the wider. This is what we should expect since in the thoracic region the musculature is lessened in amount to an extent greater than is the sensory area decreased. The dorso-ventral width of the grey commissure as taken through the central canal (column 9, tables 2, 3, 4), varies through- PRINCIPAL REGIONS OF SPINAL CORD 87 out the series. The average thickness is greater in the thoracic region than in either of the other two regions, being 0.84 mm. in the thoracic, 0.61 mm. for the cervical and 0.54 mm. for the lumbar region. It is noticeably thick in the thoracic regions of the ox, bear, hog, sheep, kangaroo, fox, Java monkey, and rabbit. Reticular formation. There is considerable variation in the amount of reticular formation as shown in the figures. This reticular network is believed to be formed, at least in part, by a dispersion of the lateral portions of the grey figure (1) by bundles of longitudinally coursing association fibers (fasciculi proprii), (2) by fibers passing out of the grey figure into the white substance and (3) by the fibers from the crossed pyramidal tracts leaving the lateral funiculus and entering the grey figure to terminate about ventral horn cells. However, the lateral pyramidal tract may have little to do with it for the animals which have their pyramidal tract in the dorsal funiculus have the reticular formation as well, often better, marked than do animals which have the pyramidal tract in the lateral funiculus. Nucleus dorsalis. A nucleus dorsalis (Clarke’s column) is well marked in the thoracic region of by far the greater number of animals here studied. In the following six mammals, kangaroo, opossum, agouti, guinea-pig, rat, and mouse the nucleus is not clearly defined. As will be mentioned, the pyramidal tract in each of these animals courses, probably in the dorsal funiculus and it may be that the dorsal position of this tract is In some way associated with the modified appearance of the nucleus dorsalis. 'These observations are based on the study of Weigert sections in which the cell-bodies are not stained, but the position and usually a very good outline of the cell-body may be seen. Proportion of grey substance to white. An idea of the relative amount of grey substance as compared with white in the spinal cord is best obtained from a study of the ratios of the absolute areas of the two. Table 7 gives such a ratio for the three reg- ions of the cord of each animal. The area of the grey figure is to the total area of the white substance as 1 is to the figure given in the table. All of the spinal cords, with the exception of 88 PEARL BRIGGS BULLARD the mouse and bat, show the lumbar region to contain the largest relative amount of grey substance, while the thoracic contains the smallest relative amount, in each animal. An average of the ratios in the three regions is valuable since it provides a com- parison of the relative amounts of white and grey substance through, what we may consider, the entire cord. The average of the ratios in the three regions emphasizes the well known fact that, in general, the smaller the animal, the greater is the pro- portion of grey substance to white. A glance at the drawings of the cervical and thoracic regions of the elephant (fig. 1, C and Th) and the corresponding regions of the bat (fig. 24, C and Th) show the relatively high proportion of grey substance in the smaller animals. TABLE 7 Ratio of area of grey substance to area of white substance. Table 7? records the ratios of grey substance to white substance obtained by dividing the figures in column 2 of table 6 into those of column 6, table 6, that is to say, the area of grey substance is to the area of white substance as 1 is to the figures recorded in columns 1, 2, and 8. ANIMAL Poe Deana a barge LSP HOMIb sie, seep: «i. wae 4.8 12.6 VOESE osha), cice eo. es. | 4.9 | 8.3 2.7 See Cs hen Rs 5 See. 3.6 6.1 2.3 4.0 [BN es Ry re 3.0 7.9 2.4 4.4 LI 2s, Ae Agee creo th ee 3.6 7.8 4.3 5.2 Man. 5.0 9.5 ss 55 SHCS) Olan A a cen a ae a Das pe: oe) 4.0 IAD GATOORE coherent oe ae. 4.3 9.8 2.6 | 5.5 ADOn ante te uh. Veta 5. 3.1 9.4 1.5 4.6 Dh ak aC 2.2 4.3 Ay Da LOPS ker eountsta Rept os Oe: See 2.8 8.5 2.2 4.5 Ch ahh aap le A eae Oe 2.6 Toil 1.5 3.9 EMER OGY surat yp acacsale + Baste hack 2.8 8.9 led, 4.4 Rhesus monkey.............. 2.0 Wee 1.6 3.6 Grom Syne Mon Seeakias a2, airs ay. Fi 5.9 1.6 3.4 OL SON oe, Se Cae eee 2.3 6.2 er 3.4 Spider monkey.............. | La 5.4 1.3 2.8 Java MONKeY 04... osc. blew. 7) 3} 6.9 0.9 3.3 PR ae Tc Ai dacs fv atheersine et 2.3 4.4 1.6 Bin PAE OUGIG ORNS se said bre Siac oes one 7.3 1.2 3.9 OIE ARI aide stun dn 22 2.9 hor’ 2.1 EMV eA Sots hui mn as antls 1.8 2.3 1.5 1.8 MIO MIRG a eon rs 5G veteran en, ell 1.6 1.2 13 (a a a cd 0.6 0.3 0.9 0.6 | | | PRINCIPAL REGIONS OF SPINAL CORD 89 White substance Dorso-intermediate sulcus. In the kangaroo, raccoon, opossum, and Java monkey, a dorso-intermediate sulcus is clearly present in all three regions (figs. 8, 18, 15 and 18). In a much larger number of species it is clearly evident only in the cervical region, man being among this number. In some animals, the ox, sheep and dog, for example, in Weigert preparations, it is not to be seen in any of the regions. In most of the sections, considerable difference is noted between the areas of white substance on either side of this sulcus. The area nearest the dorsal median septum, corresponding to the fasciculus gracilis in man, is composed of relatively small, closely packed axones which give it a darkened appearance in transverse sections stained by the Weigert method. The lateral area which corresponds to man’s fasciculus cunea- tus, is composed of relatively larger axones. Singer (’81), who describes the origin and position of the fasciculus gracilis in the dog, shows in his drawings no septum between it and the fasci- ulus cuneatus. It must be that in certain animals the factors which determine whether there shall be an ingrowth of the pial connective tissue to form this sulcus are different during fetal life while the fasciculus gracilis and fasciculus cuneatus are being acquired and becoming medullated. In most cases where the sulcus is wanting, one may observe in the cervical and thoracic regions that the fibers are smaller and more closely accumulated, and that the area is darker, near the dorsal median septum than in the more lateral areas of the dorsal funiculus. Position of pyramidal tract. Simpson (’02) describes the pyra- amidal tract for the dog, cat and monkey as situated in the dorsal part of the lateral funiculus. The guinea-pig and mouse, according to Reverly and Simpson. (710), who confirm the earlier work of Von Bechterew and Von Lenhossek, have the pyra- midal tract in the dorsal funiculus. King (’10) has traced the pyramidal tract of the rat into the dorsal funiculus. Spitzka (86) states that the sheep and ox have no fibers, to be seen macroscopically, which cross from the pyramids in the medulla oblongata into the lateral funiculus. The elephant, according 90 PEARL BRIGGS BULLARD to Hardesty (’02), has a part at least of the pyramidal tract situated on either side of the mid-line between the dorsal and ventral lamina of the grey commissure. Believing that it corre- sponds to the lateral or crossed pyramidal tract in man, he has designated it ‘fasciculus cerebro-spinalis internus’ (fig. 1, C and Th). Burkholder (04) describes this tract in the sheep as oc- cupying the same position as in the elephant and has termed it the ‘fasciculus cerebro-spinalis internus’ after Hardesty. King and Simpson (’10) state that the pyramidal fibers for the sheep are situated in the reticular formation in the lateral aspect of the dorsal horn. The specimen of sheep here studied, as well as the ox, presents in all three regions a structure identical in posi- tion. to that described by Burkholder (figs. 3 and 7, C, Th and L). Symington (’08) states that the kangaroo has the pyramidal tract in the dorsal funiculus and the rabbit has the tract in the lateral funiculus. I have compared sections through the pyramidal decussation in the medulla of the agouti and opossum with those of the rat, mouse and kangaroo, and am of the opinion that the pyramidal tract in the former animals, as well as in the latter, is situated in the dorsal funiculus. The agouti, like the rat, mouse and guinea-pig, is a rodent, while the opossum, being a marsupial, is related to the kangaroo. From an examination of sections through the medulla of the raccoon and of the fox, the pyramidal fibers appear to course in the reticular formation. However, the experimental method is the only trustworthy one for determining the position of any fiber tract. Comparison of funiculi. As is to be expected, all of the cords here considered have, in each region, a dorsal funiculus which is exceeded in actual area in transverse section by the ventro-lateral funiculus (table 6). The comparative size of the funiculi can be best expressed in the form of a ratio. Table 8 (a and b) record such ratios for each region, obtained by divid- ing the area of the ventro-lateral funiculus by that of the dorsal funiculus. It is clear that the higher the ratio, the smaller relatively is the dorsal funiculus. For example, table 8a, col- umn 4, gives man as having the lowest average ratio, 2.10, which PRINCIPAL REGIONS OF SPINAL CORD 9] means that the average size of the ventro-lateral funiculus for the three regions is 2.10 times the size of the dorsal funiculus for the three regions. The fox with the highest average ratio, 5.73, shows therefore relatively the smallest dorsal funiculus of the series. While the human cord does not show the largest relative size of the dorsal funiculus in either the cervical or the lumbar region, being surpassed in the former by the raccoon and rhesus monkey, in the latter by mouse and rhesus monkey, yet the average of the three regions places man first. In other words, all the other TABLE 8 a Ratios of dorsal funiculus to ventro-lateral funiculus. Table 8a has been computed from data in table 6, by dividing the figures in column 4 by those in column 3, table 6. In other words, the dorsal funiculus is to the ventro-lateral funiculus as 1 is to the figures in columns 1, 2 and 3 below. Column 4 records the average ratios of 1, 2 and 3. The animals are arranged according to body weight. ANIMAL ee aaa bare ar aie aaa wie i RATIO Rlephamtcee tater as acs 2.55 4.07 L: SSS ae eee 3.61 - 6.73 Deol 2.96 CD Lee Shee 3.50 6.90 see 4.54 LST ei Lae Ae Ae Saco 4.16 2.18 3.20 15 Cys Re oe 4.72 5.58 2.62 4.29 Man 2.19 2.26 1.86 2.10 NS LEG Op cia 2 eee ca ca a ee 4.27 9.04 3.51 5.60 Kangaroo...... Bin Al ee 3.04 4.82 ESOT ree eee ae 2.56 3.70 2.38 2.88 1D KO, 2 2: fen cack Rete ec eI RR Ree eee 3.24 6.41 3.24 4.29 CRS Oe One acc ee eee 4.33 8.42 4.44 5.73. IGS: veda ee auton meas Be ieee 3.15 6.71 2.51 4.12 IUSCCOOMER A Ate) W.Va ew. Sian ciek 2.13 3.17 2.92 2.74 Rhesus monkey. 2..352.2..... 2.18 7.03 1.75 3.65 PROOSSUTM Jaen we 8 sss 2.52 4.91 4.12 3.85 Cig oe eae as Oe 3.34 3.45 2.60 3.13 spider monkey 22% 6... oss) Doeves PD STIS 2.12 2.39 ava MONKEY... fos syne es: 2.88 6.75 3.94 4.52 ] SLL ee eae oe 4.30 6.10 3.98 4.79 ANOTHER Seb eee 2.99 4.35 2.84 3.39 (Oe eae a 3.59 3.58 3.58 3.58 La Shee oae, o 3.05 Sale 2.90 3.04 DURONIBE rela a PA eos ee a ks 2.34 3.03 1.67 2.34 Bat ten ok Peer Re AT. %) 2.48 2528 1.91 2.20 THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, No. 1 92 PEARL BRIGGS BULLARD cords in the series have average dorsal funiculi smaller in pro- portion to the ventro-lateral funiculi than does the human spi- nal cord. The dorsal funiculus in man is made up, in large part, of ascending axones of spinal ganglion neurones, a large propor- tion of which connect the cord with the encephalon. The latter reaches its highest development in man and the dorsal funiculus is correspondingly increased in size. We might expect the cer- vical region in man especially to show a larger ratio for the dorsal funiculus than does the thoracic or lumbar region since TABLE 86 ‘ Ratios dorsal funiculus to ventro-lateral funiculus. Table 8b records the same data as table 8a but with the animals arranged according to the relative size of the dorsal funiculus in each region of the cord instead of according to body weight. - CERVICAL THORS LUMBAR AVERAGE Baccoon. <2... =)22.13>) Man. «+. ..2263|/ Mouse sneee TESA | IMUM ere ores ec 2.10 Rhesus monkey. .2.18 | Bat.........2.27 | Rhesus.:mon- Babicicsgse.s eoeed Mant v.ta 32.62.19." Spider mon- GWE s Sts 1.76 ; Mouse.......2.34 Spider monkey... .2.32 Key:. cc. sean oe elle! 1.86 | Spider mon- Mouse... 2. ore. 72204 || Mouse: 2 sasonOSnl ea tierra OL ké6y- ee 2.39 Bat.......5.....::2:438'| Raccoon....3.17 | Spider mon- Raccoon..... 2.75 Opossum. sc eoe | RAG. 5c dae Sil Were eee vk es Le | ADC a0 oe eee 2.88 Hlephaniti. ce: POO || Cath... aaeee 3.45 | Bear. yo... 2218" | Horses ee.eee 2.95 Ape........:......2.56 | Guinea-pig.3.58 | Horse.:.....2.31 | Rat. ..3.04 Java monkey..... 2288| ADE. 2c SeTOMPADer cree. 00 | Catuat vee 3.13 POUT: oie eee 2.99 | Hlephant... 4,07 \syma.. 2.2251 "| Bear... ccsec 3.23 actrees tees: bern oe BOD 3) (pear. see AGH CGD: Byer. « es: 2.60 | Agouti.......3.39 Dynigencten: fspte SOM PAC OUbE hme ANS DUELOG eh acne 2.62 | Guinea-pig. .3.58 Dope. oferta 3.24 | Opossum. ..4.91 | Agouti.....2.84 | Rhesus mon- Gate aoe kb Dist WELOR’ sc) eee Deaomelwtitinn. ceo cea: 2.90 key 2.02... c0nee SBA Aye tes Bo va 3.00 | Rabbit... ..<. 6.10 | Raccoon....2.92 | Opossum. ...3.85 Ox ev eco BEBO Doge ute see 6.41 asengaroo . -3.04°) Lynx. . 2.5. 4.12 Guinea-pig....... Bees) ebay mies fers CHL WOR oes 4 DOR... oes cae 14.29 ELOUSGnemt a Henao Ole| EVORSGs)..0- ke efor |p Dire esa a ae Hee | PELORe ose soe 4,29 Kangaroo. .:..... 3.71 Java mon- Sheep....... 3.51 | Java mon- HEED .s unas oak Ae |) Key... on 6.75 | Guinea-pig .3.58 OY) va vi oe ee UE DRSEG Jin 9,8 ocak 4.30 | Ox..........6.90 | Java mon- Ox.5.5.. 3.95 BO Rapes tech sf af 4.33 | Rhesusmon- eV au went. 3.94 | Rabbit......4.79 TRO RGA hits £5 <8 4.72) key, .....%208)|¢Rabbig.s..... 3.98 | Kangaroo....4.82 Kangaroo. .7.72 | Opossum. ..4.12 | Sheep.......5.60 (eB OnEs ei es aes oe MAM 149 ACTED GEM PCE EVO ps Oe «eu 8 Foie, Fe Ldn RNA OEE ich og A 152 iS OUERAN TTI es cinema ee Roe OR ERE SSTA esc Ele aoe ne a 155 LON) ESTOWIGIND (BILE eee Oe Ae ee A) oS A ae ae es a 163 INTRODUCTION Balfour (78), working on elasmobranch embryos, was the first to observe that the separation of the mesodermic layers which gives rise to the body cavity of the trunk, extends for- ward into the head, and forms a cavity which later becomes 119 THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, No. 2 JANUARY, 1913 120 CHARLES EUGENE JOHNSON divided into a number of segments, called by him head cavities. These head cavities, later looked upon as true somites, became, as is well known, of considerable importance in connection with the question of the original segmentation of the vertebrate head. The bulk of the work on the head cavities has been done on fishes, especially elasmobranch embryos, and a varying number of such cavities has been found in different members of the group, nine being probably the average. The head cavities or somites lying in front of the otic region have furnished the greater inter- est, as here evidence of segmentation has been effaced to a greater degree than in the opisthotic region, where cranial nerves of the vagus group, well differentiated somites, and the arterial and gill arches have been considered by investigators as strong evi- dence of metamerism. The scarcity of metameric evidence in the prootic region is associated with the progressive develop- ment of the brain and associated sense organs, so that the higher we proceed in the vertebrate series the more obscure become the traces of any metamerism which may have existed in the pre- cursors of the group. The problem of the head somites in the Amphibia has received little attention, although Scott and Osborn as early as 1879 found that a portion of the coelom was present in the head and became segmented by the development of gill clefts; and Miss Platt (94) found sorhites in Necturus corresponding to the pro- otic somites of elasmobranchs. In the Mammalia no head ecavi- ties can be said to have been found, although Zimmermann (’99), for a human embryo of 3.4 mm., has described a number of small but clear-cut vesicles which may have been’ vestiges of such structures. For reptiles and birds on the other hand, cavities or somites corresponding to the first three head somites of elasmo- branchs have been established. These cavities or somites accord with the prootic somites of the latter group in that they occupy corresponding positions in the mesoderm, have corresponding nerve relations, and in each case give rise to corresponding mus- cles of the eye-ball. But whether a particular somite in one class of vertebrates is strictly homologous with a pare somite in another group, is of course uncertain. HEAD SOMITES AND EYE MUSCLES IN CHELYDRA 11 To morphologists looking to the problem of the segmentation of the vertebrate head, the important question is whether these so-called head somites or cavities represent true somites, compar- able to the somites of the trunk and occipital regions, and are therefore marks of a primitive segmentation of the head. The answer to this naturally depends upon each investigator’s indi- vidual conception of what constitutes a true somite; and, as Filatoff (@07) has pointed out in this connection, the variability of a somite in the head region of higher vertebrates, due to the disturbing influence of a greater development in brain and sense organs, must be taken into consideration. Some forms, for instance, may be so highly specialized that all the characteristics of a typical somite have dropped out. As will be noted in the review of literature, the question has been answered in the af- firmative by investigators of the head cavities in the Reptilia. For the Aves, Rex (’05), from extensive studies on this group, believes that these structures are products of the visceral meso- derm, and hence cannot be considered true somites, which are differentiations of the dorsal, or paraxial, mesoderm only. REVIEW OF LITERATURE THE HEAD SOMITES The first observations on the head somites of the Reptilia were made by Van Wijhe (’86). This author, in embryos of Lacerta, found what he considered to be the homologue of the first head somite of selachians, in the form of a large sac the wall of which consists of a single layer of cells, lying on each side close to the posterior surface of the optic vesicle. There was no connection between the cavities of these two sacs. Because of their position, their association with the oculomotor nerve, and the later transformation of parts of their walls into the same eye muscles that arise from the first head somite in selachians, Van Wijhe declared them homologous structures. In regard to a second head somite in reptiles, Van Wijhe makes no mention. 122 CHARLES EUGENE JOHNSON At a place corresponding precisely to the position in which the third head somite of selachians is situated, Van Wijhe found a solid mass of cells formed from indifferent embryonic mesoderm, which grows forward, becomes associated with the abducent nerve, and gives rise to the Musculus rectus lateralis. This cell-mass he accordingly calls the homologue of the third head somite of selachians. Hoffmann (’88), also working on embryos of Lacerta, (L. agilis), found that at a stage in which the optic vesicles are being formed, the first head somites are rather small cavities, one on each side, the walls of which consist of a single layer of cells. These somites are both elongated medially into processes connecting one with the other in the midline. The cavity of the somite does not continue into the process, the walls here being closely apposed. By further development these somites become greatly enlarged, and are then connected by a cross-canal (‘Quer-canal’) which, at first narrow, soon becomes extraordinarily wide. The end of the notochord lies in close contact with the posterior wall of the canal. Later the canal disappears and out of the walls of the somite are developed those eye muscles which are innervated by the nervus oculomotorius.1 Lying above the first gill cleft and just below the ganglion of the N. trigeminus, or exactly in the position where the second head somite of selachians is situated, Hoffmann found a cell-mass conspicuous on account of the peculiar arrangement of its ele- ments. The cells on the periphery are plainly arranged as an epithelium, lie in a single layer, and enclose a rather indistinct cavity, ‘‘so that quite evidently we have to look upon this cell- mass as the homologue of the second head somite.”’ A short distance posterior to the second head somite, but somewhat further medially, Hoffmann found in the same develop- mental stage, and on each side, two smaller separate and dis- tinct cell masses, in which also the cells are more or less epithelial in their arrangement, and show traces of a small enclosed cavity. 1The muscles supplied by the oculomotor, trochlear, and abducent nerves respectively, will be frequently referred to as the ‘oculomotor,’ ‘trochlear’ and ‘abducent muscles.’ HEAD SOMITES AND EYE MUSCLES IN CHELYDRA 123 Hoffmann was at a loss as to how to interpret these two masses, but he stated that it was conceivable that the anterior one cor- responded to the third head somite of selachians, the posterior mass to the fourth. As opposed to this he found that the latter did not occupy a position above the second gill pouch and below the auditory vesicle, as does the fourth head somite in selachians, but that it lies above the first gill cleft. Furthermore, in sela- chians the M. rectus lateralis is developed from the third head somite and is innervated by the N. abducens. In lizards another muscle is also innervated by this nerve, namely the M. retractor oculi. This muscle is not found in selachians. It is probable therefore, according to Hoffmann, that both cell-masses belong to the third head somite and that the anterior mass gives rise to the M. rectus lateralis and the posterior mass produces the M. retractor oculi. This he was unable to verify for lack of material. The head somites of Anguis fragilis were investigated by Oppel (90). The youngest embryo studied by him was one of 11 segments. At this stage, at the place where Hoffmann found the first head somites of Lacerta, Oppel describes his observa- tions in effect as follows: From the point where the anterior, blind end of the foregut abuts against the floor of the forebrain the mesoderm extends from the midline laterally into the head, on both sides. In front of this point there is no mesoderm. A part of the laterally extending mesoderm is noticeable as being sharply differentiated from the rest. This part grows out later- ally from the midline, gradually broadening, and extends anter- jorly toward the eye. It has the form of two wings attached at a common point. These mesodermic wings lie behind the optic vesicles, only slightly separated from them, and, curving around them laterally and ventrally, they extend still further forward. The connecting-bridge (‘Verbindungs-briicke’) of these two wing-like structures, which at the same time forms the point at which the chorda and gut-wall meet, has a posterior protrud- ing thickening or process, in which the end of the chorda disap- pears. Oppel calls this structure uniting the organs mentioned the prechordal plate (‘Praechordalplatte’). The foregut touches 124 CHARLES EUGENE JOHNSON the prechordal plate below. The mesodermal wings differ from the surrounding head mesoderm in that their cells are more densely packed. In some sections the cells in the lateral region of the wings are otherwise arranged. There appears here a small cavity, around which the cells are radially placed. Such a structure, according to Oppel, is a characteristic somite. The cell-stalk which extends from the somite to the midline, Oppel calls the ‘Stiel,’ and its length, according to him, permits the somite to lie at some distance laterally behind the optic vesi- cle. The somite portion differs from the corresponding somite found by Hoffmann in Lacerta, in that it is sharply marked off from the ‘Stiel’ or connecting-stalk. ; A short distance anterior to the auditory vesicle, at the side of the hindbrain, Oppel found the homologue of the third head somite of selachians as a mass of cells arranged radially about a small cavity. On the cranial border of this somite, seemingly growing out from it, he observed a smaller rather ‘indefinite cell-mass, which he interprets as corresponding to the anterior of the two somitic structures found at this place in Lacerta by Hoffmann. The structures here were not separate and distnct from each other as in Lacerta, and Oppel was unable to add to the suggestion as to their significance. Regarding a second head somite in Anguis, Oppel is less cer- tain. In an embryo of eleven segments, however, a short dis- tance caudad of the first head somite and somewhat nearer the midline, he found a small structure which answers the require- ments of a typical somite. Oppel’s figure shows it in secticn as having a well defined epithelial wall, one cell deep, enclosing 2 small but distinct lumen. It has no connection with any other structure, and a similar body occurs also on the other side. In an embryo of thirteen segments he found only a small heap of cells at this place, and in older specimens no further’ trace of it was found. He could establish no connection between this somite-like body and the later appearing M. obliquus superior. The Lacertilia have been investigated also by Corning (‘00). His observations were made upon embryos of Lacerta muralis and L. viridis, two forms representing essentially like conditions. HEAD SOMITES AND EYE MUSCLES IN CHELYDRA ~° 125 In an embryo of the latter species of 9 segments, two wing-like cell-masses, similar to those of Anguis fragilis, partly enclose the ventral wall of the brain tube; but no structure having somite characteristics is present. The. ‘Stiel’ or connecting-stalk of the somite, in the midline, is connected with the entoderm of the foregut, but Corning makes no mention of a prechordal plate. Later a cavity appears in the lateral part of the wing- like cell-mass, which gradually expands into a large sac with walls of cubical or even flat cells. The cavity has continued through the cellular ‘Stiel’ so that a very wide canal now connects the right and left somites. Corning evidently found no structure corresponding to the second head somite of Oppel and Hoffmann, and treats of the development of the M. obliquus superior in connection with the gill-arch musculature, as it arises, according to him, from the dorsal portion of the trigeminal muscle anlage which grows out anteriorly above the eyeball. For the third head somite this author recognized a structure which he states agrees in every respect with the third head somite of Anguis, and later gives rise to the muscles innervated by the abducent nerve. This somite, he says, is difficult to locate in younger stages, but later is easily found as a cell-mass lying close to the lateral side of the internal carotid artery and some- what medial to the trigeminal ganglion. For the Chelonia, the only work on the head somites known to the writer is Filatoff’s article (07) on Emys lutaria. Accord- ing to this author, the first head somite of Emys is developed from a mass of cells which grows out laterally from the thickened dorsal wall of the anterior end of the foregut. This thickened part of the ‘gut-wall forms at that stage the common origin of both notochord and first head somite. The middle portion of this thickening then differentiates into the chorda, the lateral portions grow outward and give rise to the first head somites. In an embryo of 18 segments the laterally lying first head somites are still connected in the midline by the cell-mass which pushes out from the intestinal wall, which Filatoff now calls the ‘Zwisch- enplatte,’ and which corresponds to the ‘Praechordalplatte’ of 126 CHARLES EUGENE JOHNSON Oppel. The end of the chorda approaches this closely, but is separated from it by an insignificant cell-mass which later be- comes more sharply differentiated from both the ‘Zwischenplatte’ and the chorda, but eventually degenerates into mesenchyma. When fully developed the first head somites in Emys are large, thin-walled ‘sacs’ connected with each other by a narrow canal resulting from the ‘Zwischenplatte.’ The second and third head somites were found differentiated in an embryo in which spiracular and first gill clefts had appeared. The second somite lies just below the developing N. trigeminus; it has a lumen and its upper or dorsal wall, especially, is formed by a distinct layer of close-set cells. The third head somite is represented by a heap of cells lying between the second somite and the auditory vesicle, and above the spiracular cleft. In this stage it possesses neither lumen nor the characteristic radi- ation of cells. Later, however, a rather indistinct radiation appears, and this is the only character, according to Filatoff, which gives this structure claim to being a true somite. A cavity is at no time developed. A compound nature of the somite such as described for the Lacertilia, was not observed in Emys. THE EYE MUSCLES Investigations of the development of the eye muscles in Rep- tilia have been fragmentary. The works of Corning (’00) and Filatoff (07) contain the most complete accounts. According to Corning the oculomotor muscles arise at definite places on the wall of the first head somite. These are chiefly the dorsal and ventral regions, while the lateral region, and the antero-medial wall which is directed towards the optic cup, take no part in the formation of the eye muscles. From the first mentioned parts muscle-forming cells grow out forming muscle ‘buds,’ and at the same time out-pocketings or folds of the wall occur, but to no great extent. The muscle buds or anlages thus formed grow out dorsally and ventrally, and then take an anterior and lateral direction towards the eye-ball. The ventral outgrowth takes the lead; it is bifureated at its anterior end, HEAD SOMITES AND EYE MUSCLES IN CHELYDRA 127 and Corning compares these two divisions with Hoffmann’s find- ings for Acanthias, and would call them respectively the Mm. obliquus inferior and rectus inferior. Both are connected with each other for some distance from their point of origin on the somite wall. The dorsal anlage has not, at this stage, advanced very far and Corning states in regard to it that he was able to establish only that it divides into parts to which the upper branch of the N. oculomotorius is given off, and that these parts give rise to the M. rectus superior and probably to part of the M. rectus medialis. The development of these muscles was followed no further by Corning. As before mentioned, the M. obliquus superior, according to this author, arises from the dorsal part of the trigeminal or max- illo-mandibular muscle anlage. On plate 6, figure 33, he pic- tures the M. obliquus superior as an uninterrupted dorsal exten- sion of the trigeminal muscle-mass, ending a short distance above the ophthalmic division of the trigeminal nerve. No structure answering to the second head somite of other authors is thus recognized. The abducent muscles are derived from the earlier mentioned third head somite. Corning does not associate the two muscles of this group with any two divisions of the third head somite, and does not give figures of the last named structure, because, he states, it agrees in every respect with the figure presented by Oppel for Anguis fragilis. From the conditions found in a late embryonic stage of L. vivipara, however, he remarks that, for the musculature innervated by the abducent nerve, one has to distinguish between two origins: a posterior one, from which proceeds the greater part of the M. retractor oculi; and an anter- ior one, from which the M. rectus lateralis arises. This is because he finds that part of the abducent muscle-mass becomes attached posteriorly to the trabeculae, and part, passing medially between the hypophysis and the trabeculae, becomes attached to the bony plate separating the hypophysis from the oral cavity. Filatoff’s observations on the oculomotor muscles agree with Corning’s, but are likewise incomplete. On plate 10, figure 28, 128 CHARLES EUGENE JOHNSON he shows a dorsal and a ventral thickening of the wall of the first head somite. From conditions shown by a much older embryo, figure 32, he concludes that the dorsal gives rise to the M. rectus superior and that the ventral one is the common anlage of the Mm. obliquus inferior, rectus inferior, and rectus medialis. The M. obliquus superior he derives from the dorsal part of the second head somite. The abducent musculature is treated by him as one mass from the time of its appearance as a single heap of cells repre- senting the third head somite, up to the advanced stage shown in figure 32, when, he states, a ‘Zweiteilung’ has taken place,: with a corresponding forking of the abducent. nerve. DESCRIPTIVE PART: MATERIAL AND METHODS The following investigation of the head somites and eye mus- cles in Chelydra grew out of a study of the mesodermic somites of the trunk region undertaken at the suggestion of Dr. B. M. Allen, while doing graduate work at the University of Wisconsin, in the summer of 1910. In continuing this study after returning to Minnesota I became interested in the somites of the head and began a more thorough study of them with the following paper as a result. A considerable part of the work on this problem including the making of the wax models was done during the past summer in the Laboratory of Comparative Anatomy of the Harvard Medi- cal School. The models were made under the guidance of Dr. Frederic T. Lewis, to whom I am indebted for many favors, helpful suggestions and never failing interest. It is also a great pleasure here to acknowledge the kind interest and encourage- ment of Dr. Minot, who generously placed at my disposal the entire Reptilian series of the Harvard Embryological Collection, which was found invaluable in checking up and verifying a num- ber of uncertain points in my own series, and for making a number of instructive comparisons. To Drs. Minot and Lewis jointly I am indebted for obtaining the services of Mr. William T. Oliver, of Lynn, Massachusetts, by whom all the drawing of the wax HEAD SOMITES AND EYE MUSCLES IN CHELYDRA 129 models were made, and also for valuable criticism and sugges- tion in the final preparation of the manuscript. The material used in this study was obtained by the aid of Prof. H. F. Nachtrieb, from the Embryological Supply Station of Mr. Albert Allen, Madison, Wisconsin. All the material had been faultlessly fixed and preserved. The following fixing agents were represented: Tellyesniczky’s bichromate-acetic; sublimate- acetic; and Zenker’s fluid. The embryos had all been preserved in 80 per cent alcohol. The specimens prepared for study were stained in toto in Meyer’s hemalum for periods varying from twenty-four to thirty- six hours. With the exception of two early series which were not counterstained all the remaining were stained on the slide in eosin. Sagittal sections proved by far the most satisfactory for the study undertaken. But these were in a number of cases supplemented by cross sections of corresponding stages. A num- ber of temporary wax reconstructions were made from time to time, of various structures, to aid in determining their form and relation to other parts. THE HEAD SOMITES The youngest Chelydra embryo studied in the preparation of this paper was a 2-mm. specimen with five segments. In the prootic region of the head the dorsal mesoderm has a compact uniform appearance on each side, becoming less dense towards its anterior limits. A careful scrutiny of the district, however, failed to reveal any differentiation of the mesoderm which might indicate a possible somite area. The next older specimen was a 3.5-mm. embryo’with ten segments, in which, as the following description will show, the mesoderm of the head presents differ- entiations, the earliest phases of which undoubtedly may be observed in stages lying between the*two here mentioned. 3.5-mm. embryo (10 segments); transverse series: figures 1 to 8 In this embryo the neural tube is still open at the anterior end. Due to the flexure of the tube the plane of section is hori- 130 CHARLES EUGENE JOHNSON zontal to the region in front of the hindbrain. In carefully exam- ining the anterior portion of this series, beginning with the first sections which are dorsal, and proceeding ventrally, there appears a section at a level slightly above the floor of the mid-brain and passing through both optic evaginations, in which a group of closely packed cells is seen lying in the mesoderm at the side of the neural tube, opposite the constriction between the dienceph- alon and the mesencephalon. Following this cell-group four sections further ventrally it appears as a well-defined, though rather small, structure in which the cells are arranged radially about a central point; their nuclei are more deeply stained than those in the surrounding mesoderm and lie toward the periph- ery. In some parts of the structure there seem to be two or three irregular layers of cells, in other parts but one. In the next section (fig. 1) there appears what seems to be a narrow slit-like cavity which can be traced through only two sections. This feature can be made out only with high power (about 300 diameters), but the entire body is readily observed with low power (65 to 80 diameters). Four sections further ventrally the limit of the structure is reached. It thus extends through a total of six 8-micron sections. Frequent mitotic figures occur throughout. Separated from this structure by not more than two sections, a second group of cells appears, smaller than the first, but show- ing in two consecutive sections a similar radial arrangement of the nuclei about a central clearer protoplasmic area, with doubt- ful traces of alumen. Beyond this group, which extends through four sections, no further differentiations in the mesoderm can be seen in this region. ‘ Through the hind-brain region, the plane of section falls at right angles to the neural tube. The notochord follows the flexure of the tube and is sharply bent so that its anterior end is seen in horizontal section, lying slightly separated from the ventral brain wall. On each side, near the ventro-lateral wall of the hind-brain and at the level of the chordal flexure, a sharply differentiated body appears in the dorsal mesoderm, which resem- bles a typical somite (fig. 2). Each of the bodies consists of a HEAD SOMITES AND EYE MUSCLES IN CHELYDRA 131 layer of tall cells, apparently two or three deep, arranged about a well formed cavity. The one on the left side is the larger and better developed of the two, but in actual size is smaller than the dorsal member of the more anterior group. The cavity can be traced through seven sections. The entire body with its lumen. is slightly compressed laterally, so that its dorso-ventral diameter is greater than the latero-medial. Its long axis is paral- lel to the long axis of the embryo, agreeing in this respect with the two components of the group first described. 175. 168 HEAD SOMITES AND EYE MUSCLES IN CHELYDRA PLATE 2 CHARLES EUGENE JOHNSON PLATE 3 EXPLANATION OF FIGURES 11 6-mm. Chelydra (second series). Sagittal section through second head somite and outer portion of abducent muscle-mass. X 175. 12 Section from same series somewhat further mediad, passing through the two divisions of the abducent muscle-mass. X 175. 13 9-mm. Chelydra. Sagittal section passing through outer wall of first head somite, showing the M. obliquus inferior. X 135. 14 10-mm. Chelydra. Sagittal section through middle of first head somite and ganglion ciliare. X 135. 170 HEAD SOMITES AND EYE MUSCLES IN CHELYDRA PLATE 3 CHARLES EUGENE JOHNSON retr. oc. a fe tri. \ e 77 [Penect: lat | » | _ 185. PLATE 4 HEAD SOMITES AND EYE MUSCLES IN CHELYDRA CHARLES EUGENE JOHNSON Oph. art. oO ° “ 7] 173 PLATE 5 EXPLANATION OF FIGURES 19 Wax plate reconstruction of right side of head of 5-mm. Chelydra serpen- tina. X 160. 174 2) HEAD SOMITES AND EYE MUSCLES IN CHELYDRA CHARLES EUGENE JOHNSON 175 PLATE 5 Op.c. div. max. man. 19 PLATE 6 EXPLANATION OF FIGURES 20 Wax plate reconstruction of right side of head of Chelydra embryo of 9-mm. X 80. 176 — ee HEAD SOMITES AND EYE MUSCLES IN CHELYDRA CHARLES EUGENE JOHNSON 177 PLATE 6 obl. inf. i ee 20 PLATE 7 EXPLANATION OF FIGURES 21a Wax plate reconstruction of head of 10-mm. embryo Chelydra, showing head somites and developing eye muscles. 50. Ir lat. rect. x tri. g. n. abd HEAD SOMITES AND EYE MUSCLES IN CHELYDRA CHARLES EUGENE JOHNSON obl. sup —— mes. Bo) 3 a 179 ac. S) rs} c PLATE 7 I. int I. int ob max. max. V retr. oc. man. man.aV VII 21a PLATE 8 EXPLANATION OF FIGURES 21b Wax plate reconstruction of the first and third head somites, with cer- tain adjacent structures, of the 10-mm. Chelydra shown in figure 21 a. Seen from the posterior side. > 100. [SO PLATE 8 HEAD SOMITES AND EYE MUSCLES IN CHELYDRA CHARLES EUGENE JOHNSON “qo “yur Wad ‘pow “Jul ‘1.8 [| *W00d Pay), “Abke}! ‘3 She) ‘dns ‘x01 ‘yaw *ydo ‘UD IA ‘u "Is OSes RE 90 *1Q04 ne Le ve sity, Sioa “Soul "yw (yedI 181] PLATE 9 EXPLANATION OF FIGURES 22 Wax plate reconstruction of the left eye-ball and adjacent structures of an embryo Chelydra of 1l-mm. Seen from the median side. 66. On account of the cephalic flexure, when the embryo is in the upright position and viewed from the side, dorsal and ventral, in figures 22 and 23, are as indicated on the plates, D.V. 182 HEAD SOMITES AND EYE MUSCLES IN CHELYDRA PLATE 9 CHARLES EUGENE JOHNSON n. abd. tri. g. NS retr. Oc. ime (exes at rect. Sup rect. = : x ——— X man. V —— oph. art. max. V obl. sup. n.oc.2 rect. it eee q s ae ON na. cil. Op. St. y —~ obl. inf. aoe rect. med. 22 183, THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO. 2 PLATE 10 EXPLANATION OF FIGURES 23 Wax plate reconstruction of the right eye-ball and adjacent structures of an embryo Chrysemys marginata of 9 mm., Harvard Embryological Collection, series 1085. Seen from the median side. X 66. 184 HEAD SOMITES AND EYE MUSCLES IN CHELYDRA ; PLATE 10 CHARLES EUGENE JOHNSON ; rect. Sup. op. st. Solis rect. inf. ‘tect. med. iN : obl. inf. 23 185 tri. g- n. abd. man. V max. V n. abd. 2. rect. | at. Glee ‘oe THE DEVELOPMENT OF THE MUCOUS MEMBRANE OF THE LARGE INTESTINE AND VERMIFORM PROCESS IN THE HUMAN EMBRYO FRANKLIN PARADISE JOHNSON From The Harvard Medical School, Boston TWENTY-NINE FIGURES The following paper is the second of a series of studies con- cerning the mucous membrane of the digestive tract. The first (Johnson 710) dealt with the development of the oesopha- gus, stomach and small intestine. The present paper is devoted entirély to the large intestine, and includes all its parts except the lower portion of the rectum. It is followed by an account of the effects of distention upon the small and large intestines of various animals. It is proposed to publish later an account of the development of the anal portion of the rectum, work upon which is nearly completed. The study of the mucous mem- brane of the digestive tract was proposed to me by Dr. F. T. Lewis in 1909, and as the work has progressed, I have received from him many valuable suggestions. The development of the large intestine is of special interest owing to the variety of pictures its mucosa presents. Relatively simple in the beginning and again in the adult as compared with other portions of the alimentary canal, the mucosa passes through a number of complicated changes before it attains its full devel- opment. To begin with, the smooth epithelial tube of the large intestine develops more or less regular longitudinal folds. These folds, as will be subsequently described, are later replaced by villi. Still later the villi themselves disappear. Meanwhile, glands are forming, and when the villi have entirely faded out, 187 188 FRANKLIN PARADISE JOHNSON the large intestine reaches its adult condition. The presence of ‘transitory’ villi has long been known, being first described by Barth in 1868. Since then they have been studied by a number of investigators, and several opinions have arisen in regard to their manner of disappearance. However, most of the former work has been done on lower animals, no one account giving a complete history of the changes that take place in the human embryo. In the present paper an attempt is made to describe in some detail the mucosa of the large intestine and vermiform process in a number of consecutive embryonic stages, and to present a series of pictures made from wax reconstructions illustrating the descriptions. The following account deals strictly with the human embryo, and is based on a number of carefully selected stages. The embryos used are arbitrarily divided into two groups;—the younger stages, which were sectioned whole; and the older stages, from which the various portions of the diges- tive tube were removed from the embryo and sectioned gepa- rately. The younger stages used were all obtained from the Harvard Embryological Collection. They are as follows: CROWN-RUMP LENGTH H.E.C. SERIES NO. FIXATION mm. 7.5 256 Zenker’s fluid 10.0 1000 Zenker’s fluid 16.0 1322 Picro-sulphurie 22.8 871 Alcohol and formalin 30.0 913 Formalin 37.0 820 Parker’s fluid 42.0 888 Zenker’s fluid The older stages were obtained from three different collec- tions. I wish here to express my thanks to Prof. C. 8. Minot, Harvard Medical School, Prof. C. M. Jackson, University of Missouri, and Prof. Franz Keibel, Freiburg i/Br., for allowing me the privilege of cutting out what portions of their embryos I desired for my work. The list of older stages used is as follows: DEVELOPMENT OF THE LARGE INTESTINE 189 CROWN-RUMP LENGTH FIXATION COLLECTION SERIES NO. Te eee Coe Mere 50 Zenker’s fluid Keibel 55 Alcohol Minot 249 58 Zenker’s fluid Keibel 65 Zenker’s fluid Keibel | 70 Alcohol Keibel | 73 Picro-sulphuric Minot | 116 75 Alcohol Minot 110 88 Formalin Jackson 137 99 Alcohol Minot 340 110 Formalin Jackson | 143 120 Zenker’s fluid Minot 342 140 Formalin Jackson 263 170 Formalin Jackson 222 187 Formalin Minot | 315 190 Formalin Jackson 200 Formalin Jackson 89 240 Miiller’s fluid Minot | 186 320 Formalin! Jackson | 16 Bie ela eeees toe, Ae eee Zenker’s fluid Minot | 345 Birthigeeey arc. sents acres Zenker’s fluid Minot | 341 Two weeks child‘... Zenker’s fluid Minot 1 First injected with a mixture of phenol, alcohol, glycerine, and formalin 2 Premature birth at seven (?) months. Lived thirty minutes 3 Normal fetus at birth 4 Premature at seven months. Lived two weeks THE LARGE INTESTINE Early development In an embryo of 7.5 mm. the large intestine, like the oeso- phagus, stomach, and small intestine of the same embryo, is a simple tube of epithelium surrounded by mesenchyma. It is continuous, without demarction, with the small intestine above, and with the urogenital sinus below. Its cephalic end is indis- tinctly indicated by a slight swelling, which is regarded by Lewis (11) as the beginning of the vermiform process. This swelling, which I will designate by the term ‘colic ampulla’ (ampulla coli), is spindle-shaped and has a diameter (measured from side to side in its widest place) of 0.07 mm. Below the swelling the tube becomes markedly narrower. In its narrowest portion it 190 FRANKLIN PARADISE JOHNSON measures but 0.04 mm. in diameter. As the urogenital sinus is approached the epithelial tube again increases in size and becomes compressed from side to side. This enlargement passes insensibly over into the epithelium of the cloaca. The walls of the epithelial tube also vary in thickness. Above, where it is broadest, the walls have a thickness of about 0.028 mm. and are composed of cells which have no distinct cell boun- daries. - Three rows of oval nuclei are discernible. In the nar- rower middle portion the walls average about 0.017 mm. in thickness, and show only two rows of nuclei. Where the tube is expanded in the cloacal region, the epithelium is approximately of the same thickness as in the colic ampulla. The lumen in most places is a narrow slit-like cleft, larger in the extremities than in the mid-region of the large intestine, but everywhere present and patent. It communicates with the larger lumen of the cloaca, but cannot be traced through this to the outside because of the presence of the cloacal membrane. In an embryo of 10 mm. the large intestine presents practi- cally the same relations, but shows a marked increase in size. The colic ampulla, which is now situated well out in the coelom of the umbilical cord, measures alout 0.33 mm. in diameter, and has an epithelium 0.045 mm. in thickness. A slight bud- like protuberance of almost the same size as the swelling itself, arises from it, and extends into the mesenchyma. Followed caudally, the epithelial tube of the large intestine quickly dimin- ishes in size, and continues of small size until the region of the rectum is reached. Here it presents another spindle-shaped swelling. This swelling is connected with the cloaca by a short and narrow tube. The upper narrow portion of the epithelial tube measures about 0.07 mm. in diameter, and its wall is 0.028 mm. thick. The swelling is 0.12 mm. in diameter, and has a wall thickness of 0.036 mm. The lumen is continuous as far as the cloacal membrane where it is closed off from the exterior. At 16 mm. the colic ampulla is similar in form but larger than in the preceding stages, being now 0.17 mm. in diameter. It is directly continued into the vermiform process, which is found DEVELOPMENT OF THE LARGE INTESTINE 191 in the umbilical cord, pointing away from theembryo. Large at its base where it joins the colic ampulla, the vermiform process tapers gradually towards its blind end. The before-mentioned narrow portion of the large intestine, now 0.09 mm. in diame- ter, has increased much in length. The lumen of the lower end of the digestive tube no longer leads into the cloaca, but opens to the outside by an extremely small aperture. In the further course of its development, the swelling in the rectal region becomes much larger, and longitudinal folds make their appearance. These longitudinal folds increase in numbers, and are markedly constant in position in all the older stages. Just what is the fate of these folds I am unable at the present time to state precisely, but it is not improbable that they give rise to the rectal columns (columnae rectales Morgagni) while the spaces between them no doubt develop into the rectal sinuses (sinus rectales). A discussion of the further development of this portion of the digestive tract, however, has been omitted from the present paper. The development of longitudinal folds In an embryo of 22.8 mm. one sees for the first time a change in the form of the epithelium. In the colic ampulla, which now has a diameter of 0.20 mm., the epithelium shows three low longitudinal ridges on its inner surface. These ridges also extend for short distances into the colon and vermiform process. It becomes necessary at this point to explain the manner in which the terms ‘ridges’ and ‘folds’ have been used through- . out the remainder of this article. The term ‘ridge’ has been employed to designate a thickening of the epithelium which projects into the lumen. It must have no corresponding inden- tation on its under surface into which mesenchyma would ex- tend. By a ‘fold’ is meant a projection with an indented basal surface, into which the underlying mesenchyma protrudes. This distinction is desirable, as its usage makes it possible to explain in few words the shape of the basal surface of the epithelium along with that of its free surface. 192 FRANKLIN PARADISE JOHNSON The epithelium in the embryo under consideration (22.8 mm.) varies in thickness in different regions. In the colic ampulla and vermiform process it is 0.056 mm. thick and shows three to four rows of nuclei. In the remainder of the colon down to the rectal ampulla, it is only 0.034 mm. in thickness and shows but two to three rows of nuclei. At no place are the bounda- ries between the cells distinct, but the free and basal surfaces are well marked. In an embryo of 30 mm. the whole of the vermiform process and the valve of the colon lie in the coelom of the umbilical cord. An examination of the interior of the colon in this region shows the beginnings of two to three epithelial ridges. These vary in height, the epithelium being 0.070 mm. thick, measured through the summit of the largest ridge, while in the depressions between them, it is only about 0.028 mm. thick. ‘Three or four rows of nuclei can be made out. The remainder of the colon has an average diameter of 0.15 mm. Throughout its great length the epithelial tube is still cylindrical in shape, having a wall 0.048 mm. in thickness. Three or four rows of nuclei are present. At 37 mm. the whole of the vermiform process still lies in the umbilical cord. The epithelial ridges are higher than in the former stages. In the base of the vermiform process there are four of these, in the middle only three; in the beginning of the colon, three or four. The highest ridge measures about 0.10 mm. in height and has three or four rows of nuclei. Between the ridges the epithelium is only 0.042 mm. thick and shows but two or three nuclear layers. The remainder of the large intestine gradually decreases in size when followed caudally. © The portion which is to become the ascending colon is 0.27 mm. in diameter; the transverse 0.22 mm.; and the descending colon 0.18 mm. The epithelium is about 0.056 mm. thick in all these portions. A few vacuoles, such as have been found in the epi- thelial walls of the stomach and oesophagus are present in the colon of this embryo. In an embryo of 42 mm. the ridges of the epithelium are in part replaced by true longitudinal folds. In the vermiform proc- DEVELOPMENT OF THE LARGE INTESTINE 193 ess and ascending colon three to four of these are present; in the transverse colon two to three; in the greater part of the descend- ing colon three; while in the remainder of the descending colon five to six more irregular ones. These folds vary in height from 0.014 to 0.028 mm. The epithelium is thicker on their crests than between them. It presents an appearance which is largely in accordance with a condition which Patzelt (83) has found in the large intestine of the cat embryo. He describes two types of cells. In the corners of the star-shaped lumen the cells are short and broad, and have basal nuclei which stain intensely with haemotoxylin. The cells of the second type are found on the tops of the folds. They are longer, finely granular, and somewhat denser. Their nuclei are long-oval or drop-shaped and stain more intensely than those of the first type. The for- mer groups of cells he states are the first anlagen of the Lieber- kiihn glands; the latter of the villi. The epithelium of the large intestine of the embryo under consideration (42 mm.) has been . described and pictured by Lewis (711). The two types of cells are found arranged in separate groups, but, however, are not as distinct as those of the cat described by Patzelt. In the ascending colon of an embryo of 50 mm., the epithelial tube has a diameter of about 0.23 mm., and shows four distinct longitudinal folds. These are, as shown in figure 12, rounded on their tops, and are of different heights, the largest measuring about 0.06 mm. In the piece of ascending colon sectioned for study, which measures about 0.7 mm. in length, the epithelial _ tube changes but little in shape, the four distinct longitudinal folds running throughout. The epithelium, which has an aver- — age thickness of 0.050 mm., is columnar, and, as seen in sections ten microns thick, is apparently stratified, being composed of two or perhaps three, layers of cells. The nuclei, which are oval in shape, are all placed in a zone midway between the free and basal surfaces of the epithelium, there being a clear zone of protoplasm on either side. A definite cuticular border is everywhere present on the free surface of the epithelium. Two distinct types of cells are not visible. Outside of the epithelium is a zone of loose mesenchyma which is bounded by a thin layer 194 FRANKLIN PARADISE JOHNSON of myoblasts, the circular layer of the muscularis. This is in turn bounded by a layer of mesenchyma and surrounding the whole, except at its mesenteric attachment, a distinct serous epithelium is seen. In passing ab-orally from the ascending colon into the trans- verse colon, one of the four longitudinal folds just described drops out, while a second becomes so much reduced in size that it is scarcely recognizable as a fold (figs. 1 and 13). The two remaining folds are distinct and about 0.06 mm. in height. The epithelial tube has a diameter of about 0.27 mm. The remain- ing features of this portion of the large intestine are similar to those of the ascending colon. First appearance of goblet cells In the iliac colon (50 mm. embryo), the epithelial tube as a whole is flattened from side to side. Its greater diameter is 0.36 mm., its lesser 0.18 mm. A considerable change in the condition of folds is evident. They are shallow, irregular, and more numerous than in the ascending and transverse colons. A model of a small portion of this region is shown in figure 14. The distinction between epithelial ridges and folds is here appar- ent—only those protuberances, which have indented basal sur- faces into which the mesenchyma extends, being considered as true folds. Measured’ through the ridges the epithelium is in places 0.084 mm. thick, while in the clefts between them, it is only 0.028 mm. thick. The two types of cells described by Lewis in the 42 mm. stage are distinct. A few cells on the ridges have a protoplasm which is clearer than others, and are shaped somewhat like goblet cells. Because these cells in the next few stages take on more and more the appearance of goblet cells until their identity cannot be doubted, I believe them to be goblet cells in a very early stage of differentiation. Voigt. (99) was able to distinguish goblet cells first in the rectum of a human embryo of 70 mm. The ascending colon of an embryo of 55 mm. has a diameter of 0.45 mm. Ten to twelve longitudinal ridges are found, but DEVELOPMENT OF THE LARGE INTESTINE 195 distinct folds are absent. The ridges are irregular in form and of varying size, the largest being about 0.10 mm. in height. The two types of cells are distinct now in this region of the large intestine, some of which are like those which Patzelt has de- scribed as drop-shaped. In many places large vacuoles similar to those described above are found in the epithelium of the ridges. The epithelial tube of the transverse colon is of the same size as the ascending, and shows six well marked projections into the lumen, two of which are folds. In the upper part of the descending colon, two ridges and two folds are present. In the iliac colon the folds drop out and only ridges are found. When Fig. 1! Cross section of the transverse colon of a human embryo of 50 mm. x 60. followed downward, the descending colon shows more and more ridges and when the sigmoid colon is reached there are as many as ten or twelve. Still more caudally the rectum shows folds which have taken the place of the ridges. In the lower part of the rectum, just above the rectal ampulla, practically all the ridges have been replaced by folds, varying from ten to four- teen in number. The appearance obtained from cross sections, therefore, is somewhat similar to that found in the stomach of the same and slightly older embryos—the clefts in between the ‘ridges corresponding to the gastric pits. The clefts, however, are broader and the cells of the epithelium lining them are more 1Tn this and all remaining text figures certain histological details have been omitted. 196 FRANKLIN PARADISE JOHNSON eolumnar in form than those of the gastric pits. Throughout all these portions of the large intestine the cells on the crests of the ridges differ from those between them. In the rectum the epithelium is distinctly one-layered. On the crests of its folds it presents a number of cells with clear protoplasm and basal nuclei. These presumably are developing goblet cells. In the colon of an embryo of 58 mm. the epithelial tube is found to be quite similar to that of the 55 mm. embryo just described. It is slightly smaller throughout than in the previous stage, which difference may in part be accounted for by the different kinds of preserving fluids used. In the cephalic end Fig. 2 Cross section of the transverse colon of a human embryo of 58 mm. x 60. of the ascending colon the epithelial tube has now a diameter of 0.38 mm. and shows numerous ridges and folds as seen in figure 15. More caudally in the ascending colon the epithelium is not so irregular as near the caecum. As seen in sections three folds and one ridge are present. Figure 16 shows a model of this portion of the intestine. It is to be noted that the tops of the longitudinal folds are irregular in form. ‘The diameter of this region of the gut is about 0.86 mm., while the epithelium averages about 0.050 mm. in thickness. The transverse colon of an embryo of 58 mm. shows six dis- tinct folds, as seen in figure 2. A model of one half of the tube of this region is represented in figure 17. The diameter of the DEVELOPMENT OF THE LARGE INTESTINE 197 epithelial tube averages 0.88 mm. The descending colon (fig. 18) has a diameter of 0.34 mm., is more rounded in shape, being quite similar to the more cephalic part of the ascending colon. The crests of the folds and ridges are, however, not so angular. First appearance of villr In the rectum the epithelial folds have increased in size and give to the lumen a very irregular form. As shown in figure 19, some of the folds run almost transversely. The presence of transverse folds have been noted in the lower portion of the rectum in a number of older embryos as well. Besides folds, here and there are present conical-shaped projections of the epithelium. These represent the first transitory villi of the large intestine. In a number of places the folds seem to be fused together at their tops, shutting off small rounded spaces. These spaces I have determined from serial sections to be epithelial cysts. They are found in corresponding portions of the rectum of other embryos, but are confined to this region of the large intestine alone. A portion of one of these cysts is shown in figure 19 at x. They are described in detail below. At this point it seems advisable to make the following sum- mary regarding the development of ridges and folds. In the beginning the epithelial tube is cylindrical in shape. The first changes that take place in its form are found in the rectum, where it shows a number of longitudinal ridges. These ridges are the forerunners of folds, for everywhere they later appear as if pushed in from behind by the underlying mesenchyma. Soon afterward ridges and folds are found in the descending colon, the direction of growth being from below upward. How- ever, before these changes have extended into the transverse colon, similar changes are found to be occurring in the ascend- ing colon near to the colic valve. The direction of growth here is opposite that in the descending colon, that is, ab-orally. The transverse colon is, therefore, the last portion of the large intes- tine to develop folds. Similarly, in a few of the subsequent stages, the transverse colon shows a slight retardation in the 198 FRANKLIN PARADISE JOHNSON development of vili and glands as compared with the rectum, descending and ascending colons. However, this retardation is soon overcome by an increased rate in growth, and then con- ditions found in all parts of the large intestine are quite similar. As a matter of convenience and simplicity, the development of the transverse colon has been described most completely in the remainder of this article, and other portions of the large intes- tine are described, as far as is possible, from a comparative point of view with respect to it. Fig. 3 Cross section of the transverse colon of a human embryo of 65 mm. x 60. The transverse colon of an embryo of 65 mm. differs con- siderably from that of the embryo just described. The epithe- lial tube is circular in section and measures 0.54 mm. in diameter. In transverse section (fig. 3) eighteen to twenty-three projec- tions are seen extending into the lumen. When modelled these projections are seen to be longitudinal folds and villi as shown in figure 20. The villi are everywhere arranged in longitudinal DEVELOPMENT OF THE LARGE INTESTINE 199 rows, suggesting that the folds have become broken up into segments. The folds and villi measure from 0.08 to 0.11 mm. in height and are usually between 0.07 and 0.11 mm. in width at their bases. The epithelium on their tops is distinctly sim- ple columnar in form, and is reduced to 0.019 mm. in thickness; between the folds it is 0.081 mm. thick. Numerous villi are found in the ascending colon. A compari- son of these with those villi in the lower part of the ileum shows that the two are quite similar in form and size. At a short dis- tance from the colic valve, the epithelial walls of the ascending colon become pushed in by three large mesenchymal folds, re- ducing the lumen to a narrow Y-shaped cleft. Here are found folds and villi resembling those shown in figure 20. The de- scending colon is smaller in diameter than the transverse, being only 0.45 mm. An examination of its inner surface (fig. 21), shows folds and villi, and what apparently are partially formed vili, about eight to nine rows in all. These are longer than those of the transverse colon, 0.17 to 0.22 mm., but of about the same width. In the sigmoid colon is found a condition comparable to that of the descending colon. However, goblet cells are far more numerous. First appearance of intestinal glands The epithelial tube of the upper portion of the rectum (embryo of 65 mm.) is flattened from side to side, and measures 1.17 by 0.77 mm. in cross section. A very different appearance is presented from that of the transverse colon. The epithelial wall is bent into a number of folds which are closely packed to- gether. Many of these measure as much as 0.27 and 0.36 mm. in height. The bottoms of the spaces between these projections are developing glands. Where they are cut obliquely or in cross section their basal ends are seen to be tubular in form and provided with small round lumina. Epithelial cysts are more numerous than in the preceding embryo. They represent glands and intervillous spaces which have become closed over at their tops. They show evidences THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, No. 2 200 FRANKLIN PARADISE JOHNSON of internal pressure by their bulbous appearance and by the flattening of the lining epithelium of the more superficially lying part of the cyst (fig. 11). In many respects these cysts are similar to those found in the vermiform process (compare fig. 11 with figs. 9 and 10) but differ from them by their more super- ficial position and in that they can rarely be considered to be entirely separated from the surface epithelium. Moreover, they have a different fate from those of the vermiform process. In- stead of the epithelium entirely degenerating, the cyst collaps- ing, and finally being absorbed, the cysts of the rectum open up with the intestinal lumen and become glands again, at least this interpretation seems justifiable, since the cysts gradually disappear without showing such degenerative processes as are easily recognizable in those of the vermiform process. The condition found in the transverse colon of an embryo of 70 mm. is not much in advance of the same portion of the large intestine at 65 mm. Its epithelial tube has a diameter of 0.54 mm. The lumen is relatively large and the villi project into it 0.09 to 0.18 mm. The cells forming the epithelium are tall columnar, 0.025 in height, and contain at their basal ends, elongated nuclei. The protoplasm, which stains decidedly yel- low with orange-G, appears to be mucous in character. Here and there swollen goblet cells are seen. A small portion of the sigmoid colon presents an appearance similar to that described in the rectum at 65 mm. It measures 0.54 mm. by 0.72 mm., and contains folds and villi 0.23 to 0.27 mm. in height. Epi- thelial glands and cysts are found in large numbers. In the rectum the same conditions are presented, although the epi- thelial tube is larger and the villi taller (0.25 to 0.832 mm.). The latter seem, however, to be so fused together that they appear in many places as irregular running folds. Goblet cells are everywhere numerous. In a well preserved transverse colon of an embryo of 73 mm. the epithelial tube measures 0.58 mm. in diameter. The villi and folds, some of which are now 0.22 to 0.23 mm. high, decrease to a marked extent the size of the lumen. In width the villi show a slight increase, being 0.09 to 0.18 mm. through at their DEVELOPMENT OF THE LARGE INTESTINE 201 bases. The tops of the villi are in many places so closely approxi- mated that it is quite impossible from cross sections of this stage alone to determine whether an actual fusion has or has not taken place. Because of this condition, which I believe to have been brought about by a strong contraction of the muscularis, an attempt to model these villi accurately proved fruitless. From the conditions found in the large intestine, of other embryos of about the same age, however, it would not seem probable that such a thing as an actual fusion had taken place. The epithelium on the tops of the projections is distinctly one-layered and 0.025 to 0.028 mm. thick, while between them it appears Fig. 4 Cross section of the transverse colon of a human embryo of 88 mm. x 60. two-layered, and is almost twice as thick, 0.042 to 0.052. Only the portions of the epithelium in between villi are provided with distinct basement membranes. In the descending colon practically the same conditions are repeated, with the exception that a few more villi are present. In the transverse colon at 75 mm., even though considerable shrinkage is present, the villi are seen distinctly separated from one another. Other portions of the large intestine from the two last-mentioned embryos were not obtained. In the transverse colon of an embryo of 88 mm. (fig. 4), are found numerous villi, which are arranged so that they form longitudinal rows. The epithelium on the tops and sides of the villi is similar to that of the preceding stages, being simple colum- nar in form and containing goblet cells. Between the villi the cells are tall cylindrical and conical in shape, contain oval nuclei 202 FRANKLIN PARADISE JOHNSON which seem to be closely crowded together, and stain inten- sively. These groups of cells form small knob-like projections and are the beginnings of the intestinal glands. In the ascending, descending, and sigmoid colons, as seen from transverse and longitudinal sections, both villi and the beginnings of glands are distinguishable. The villi gradually increase in length as the large intestine is followed caudally; thus in the ascending colon they are about 0.14 to 0.16 mm. in height; in the transverse, 0.18 to 0.20 mm.; in the descending colon, 0.22 to 0.25 mm.; in the sigmoid, 0.27 to 0.82 mm.; while in the rectum, 0.27 to 0.36 mm. In many places their apices are in close contact with each other, appearing as though fused. Likewise, the glands show a more advanced stage of growth as the large intestine is followed downward. In the ascending colon they are scarcely visible; in the rectum they are very dis- tinct. Except for this more advanced stage of development, conditions in the rectum are not so strikingly different from those in the remainder of the colon as at a former period. The epithelial cysts, while not so numerous, have not entirely dis- appeared. ‘Those few which remain are smaller and are confined to the lower part of the rectum. In an embryo of 99 mm. the transverse colon shows numerous villi arranged in rows, 20 to 25 in number. As seen in figure 23, few distinct folds are present, but these do not occur in any definite relation to the villi, that is, the rows of villi and the folds are not alternately placed around the wall of the intestine. From this and from what I have seen in other embryos, it seems improbable that the new villi, which are now arising at a very rapid rate, are preceded by folds. More probably they develop after the manner of the villi in the small intestine, as separate growths between the villi already formed. Numerous gland buds are also present in the specimen as shown in figure 23. Where the glands are cut in cross section, they show small but distinct lumina, surrounded by columnar cells of the mucous variety, many of which are goblet cells. On the tops and sides of the villi the epithelial cells are 0.022 to 0.028 mm. in height while in the glands they are 0.034 to 0.042 DEVELOPMENT OF THE LARGE INTESTINE 203 mm. Although shrunken from the underlying mesenchyma, the epithelial tube has increased to 1.03 mm. in diameter. The sigmoid colon has a diameter of 0.95 mm. and an epithelium which is similar in variety and of equal thickness to that just described. From this point on, measurements taken of the glands and villi can only be considered approximately accurate. This is due to the fact that there is no sharp line of demarcation between gland and villus, consequently one is unable to determine just where the gland begins and where the villus leaves off. A simi- lar difficulty was met with in the case of the small intestine. It is, however, possible, with the aid of models for comparison, Fig. 5 Cross section of the transverse colon of a human embryo of 110 mm. x 60. to judge the line of division approximately. In the following account of the growth of glands and villi, the figures given are the average of a number of measurements made from cross or longitudinal sections, or in some cases, from both. A more accurate method, which was employed whenever possible, was the direct measurement of these structures from the models themselves. In the transverse colon of the embryo under de- scription (99 mm.), the glands may be regarded as about 0.07 mm. long and 0.056 mm. broad, while the villi as 0.27 mm. tall and 0.10 mm. broad. In the sigmoid colon the glands aver- age 0.10 mm. and the villi 0.31 mm. in length. 204 FRANKLIN PARADISE JOHNSON In the transverse colon of an embryo of 110 mm. (fig. 5), the villi are long and narrow, and give to the colon the appearance of a small intestine of a slightly older embryo. Some of them measure as much as 0.36 to 0.45 mm. in length and average about 0.09 mm. in diameter at their bases. They are covered by a low columnar epithelium (cuboidal in places), which is rather poor in goblet cells. The glands are longer than those of the preceding stage, being about 0.13 to 0.16 mm. in length. The cells which line the glands are distinctly columnar, meas- uring from 0.022 to 0.028 mm. in height. By far the majority of these are goblet cells. Their nuclei are basally placed and closely crowded together, making this region of the gland very deeply stained. In the ascending colon, a similar picture is obtained. ‘The villi are, however, somewhat shorter (0.27 to 0.36) mm. Notice- able again is the greater distribution of goblet cells in the glands than on the villi, and the difference in the height of the epithe- lium in the two regions. In the sigmoid colon and the rectum, the epithelial tube is larger and flattened from side to side. The villi and glands are quite similar as regards size, shape and struc- ture to those in the ascending colon. No epithelial cysts were found in the piece of rectum examined, which was a portion taken rather high up. In a well preserved embryo of 120 mm., the transverse colon has a diameter of about 1.08 mm. in contrast to 1.26 mm. in the preceding embryo. The villi, which are closely packed together, are also shorter (0.18 to 0.27 mm.) than those of the former embryo, but the glands are of about the same length. This difference in size is probably due in part to the different preservating fluids used on the two embryos. The epithelium of both villi and glands is in excellent state of preservation and the goblet cells, which have taken the stain (orange-G) very strongly, stand out in marked contrast to the remaining cells. It is easily seen, therefore, that the goblet cells are more numer- ous on the sides than on the tops of the villi and most numerous in the glands. In many glands these cells appear to be exclusively present. Although the glands are still only short DEVELOPMENT OF THE LARGE INTESTINE 205 knoblike projections (0.11 to 0.14 mm. in length and 0.09 mm. in width), some of them appear to be double. As will be subsequently described, this bifureating of the glands is a very important factor in their multiplication. Conditions in the ascending colon, as regards villi and glands, are sunilar to those in the transverse. In the rectum the epi- thelial tube is again flattened laterally measuring 2.12 mm. by 0.69 mm. in cross section. The mucous membrane is thicker (0.32 to 0.36 mm.) than that of the transverse colon (0.27 to 0.32 mm.). Its villi are from 0.22 to 0.27 mm. in height and its glands about the same as those found in the transverse colon (0.13 to 0.16 mm.). Goblet cells are again very numerous. A few epithelial cysts were found in the extreme lower part of the rectum, but these were almost insignificant in size as com- pared to those of the former stages. Disappearance of villi In an embryo of 140 mm. the transverse colon has a diameter of about 1.7 mm. and presents from thirty-six to forty longi- tudinal rows of villi. It is apparent, therefore, that the villi are still increasing in numbers as the intestine is increasing in size. Since no distinct longitudinal folds can be found in either models or longitudinal sections, these additional villi must de- velop from the beginning as separate growths in the spaces between those villi already present. The form of the villi is shown in figure 24. Some of the tallest measure only 0.25 mm. in height. Disregarding the embryo of 120 mm., because of the undoubted shrinkage of all its parts, and referring to the 110 mm. stage, it is seen that the villi are shorter in the older embryo. Moreover, some of the villi are so short at 140 mm. that the term villus is scarcely applicable to them, but whether these are newly developed villi or dwindled-down old ones, I am unable to determine. Although the intestinal glands vary in length (0.13 to 0.18 mm.) they are on the whole slightly more advanced than before and many show signs of bifureating. From the above observations it is evident that, as the glands are in- 206 FRANKLIN PARADISE JOHNSON creasing in length, the villi are decreasing in height. In the subsequent stages of development, the villi become always lower and lower, so that it is now possible to say that the transitory villi reach their maximum height in embryos between 110 mm. and 140 mm., probably between 110 mm. and 120 mm. In that portion of the ascending colon adjacent to the colic valve, shorter villi are found (0.14 to 0.18 mm. in height), which are also lower than those in the ileum (0.27 to 0.86 mm.). Higher up in the ascending colon the villi are of about the same size, while the glands are 0.13 to 0.18 mm. in length, and in the begin- ning of the ascending colon, a few enlarged glands, such as are present in the vermiform process, are found. As regards villi and glands, the descending colon is quite similar to the trans- verse colon. In the sigmoid colon the villi are longer (0.18 to 0.27 mm.); in the rectum (0.18 to 0.36 mm.). Everywhere, however, the glands remain about the same length as those of the transverse colon. The epithelium is also quite similar throughout the whole colon. It is high columnar in the glands, lower on the sides, and lowest on the apices of the villi. Goblet cells are extremely numerous everywhere, being more abundant in the glands than on the villi. Effects of distention caused by a storing up of meconium A marked difference in the thickness of the mucosa is found between the transverse colon of an embryo of 187 mm. and that of the same portion of the intestine of an embryo of 190 mm. (Compare figs. 6 and 7). That of the former is 0.36 mm. in thickness, while that of the latter is only 0.16 to 0.20 mm., the first being about twice the thickness of the second. The ques- tion arises, how is this variation to be accounted for? It is to be noted that the portion of intestine of the 190 mm. embryo examined was filled with meconium, thereby extending its walls and increasing the size of its lumen. The total diameter of its epithelial tube is 4.2 mm., in comparison to 2.3 mm. in the 187 mm. stage. These measurements show only that the epi- thelial tube is extended in the older stage, but they do not show DEVELOPMENT OF THE LARGE INTESTINE 207 Fig. 6 Longitudinal section of the transverse colon of a human embryo of 187 mm. 60. Shows state of normal contraction (compare villi and glands with those of fig. 7). Fig. 7 Cross section of the transverse colon of a human embryo of 190 mm. x 60 (compare with fig. 6). that the circumference of one is greater in one than in the other, because in the younger stage the epithelial wall is thrown into three or four large folds. In order to determine accurately whether the folds alone would compensate for so great a differ- ence in diameter, enlarged camera drawings were made, and the length of the lines at the bases of the glands (the line at which the muscularis is just beginning to appear) was measured. The distended gut (190 mm. stage) measured 15.3 mm., while the contracted one only 11.3 mm., showing still a considerable un- accountable difference. From what I have seen in this and other sections, in the small as well as in the large intestine, I have come to the conclusion that wherever the embryonic intes- tine is greatly distended with meconium, as is often found to be the case in the older stages, the thickness of the mucosa 208 FRANKLIN PARADISE JOHNSON becomes greatly reduced. In other words, where a considerable amount of meconium is found in the intestine, the thickness of its mucosa varies indirectly with the amount of distension. Since the perimeters of the two colons under discussion were unequal in length, it is of interest to compare the number of glands present in them. This was done by counting the num- ber of glands which were practically in contact with the develop- ing muscularis mucosae. This method would not have given comparable results had not the thickness of the sections in both cases been the same (8 microns). In the distended intestine variations from 79 to 96 and an average of 86 were obtained; in the non-distended piece, a variation from 81 to 91 and an average of 85.2, showing that the number of glands is approxi- mately the same. Whether the number of villi around the intestinal wall is the same in both of the intestines, is a harder problem to determine, owing to their greater size and irregu- larity in height, and to the fact that one is given no distinct basal line, as in the case of the glands, from which to measure. Such a problem could only be determined with any degree of accuracy by making a number of models of comparatively large areas. However, from cross sections alone, it is possible to make out that in the distended intestine the villi are further apart. In the spreading or stretching out of the mucosa, both the villi and glands become reduced in length and broadened. ‘The following measurements have been made to show this: HEIGHT OF WIDTH OF . : a : LENGTH OF GLANDS, WIDTH OF GLANDS TALLEST VILLI BASES OF VILLI mm. mim. mm. mim, Non-distended. . 0.18-0.27 0.07—-0.09 0.22 0.05-0.09 Distended.......| 0.00-0.09 | 0.14-0.18 0.11-0.14 0.09-0. 14 It must be noted that the villi had in many places practically disappeared. The outer layers of the intestine are also reduced in thickness by distension, a fact which is of common observance in the digestive tube of the adult. In the non-distended transverse DEVELOPMENT OF THE LARGE INTESTINE 209 colon the submucosa, the muscularis, and the serosa together measure about 0.24 mm., the distended one, 0.13 mm. From the above given figures, the effects of distention, caused by a storing up of meconium in the large intestine may be enu- merated as follows: (1) an increase in the diameter of the epi- thelial tube; (2) an increase in the actual surface on which the basal ends of the glands he; (3) a decrease in the thickness of the mucous membrane as a whole; (4) a decrease in the length of the glands and an increase in their width; (5) a more marked decrease in the height of the villi and an increase in their width (6) a spreading apart of the glands and of the villi; and (7) a decrease in thickness in the outer coats of the intestine. The above results led to the question as to what effect dis- tention would have upon the intestine of an adult. The small intestine of a guinea-pig was experimentally distended with normal salt solution and fixed and hardened in that condition. Although the results of this and other experiments may be found in the following paper, it may be said here, that the results obtained are largely in accordance with those just described in the meconium-filled intestine of the human embryo. Because of the above described effects of marked distention of the large intestine, it has been considered necessary, for the sake of comparison, to select only non-distended portions of the colon for modelling. In the case of the embryo of 190 mm. the sigmoid colon was chosen, for while this region contains meconium, there is no distention. This is made evident by the presence of longitudinal or oblique folds of the mucous mem- brane, and by the following measurements as compared with those of the transverse colon of the same embryo: diameter of intestine 3.4 mm.; thickness of mucous membrane 0.34 mm.; thickness of outer intestinal coats, 0.23 mm. Figure 22 shows the epithelium of this region of the intestine in surface view. The villi have dwindled down until they appear merely as irregu- lar knobs which are joined together at their bases in the form of ridges. These ridges are of various lengths but all are of about the same thickness. They run in different directions and anastomose with one another, thus marking the surface of the 210 FRANKLIN PARADISE JOHNSON epithelium up into an irregular network such as Langer (’87) has described in connection with the developing colic valve. The clefts in between the ridges are deep, and into them open the lumina of the intestinal glands. An examination of the basal surface of another model (not figured), from the same por- tion of the large intestine, shows numerous tubular glands, many of which are unbranched, but the branched type is not uncommon. As stated before, the first beginnings of the intestinal glands appear as small knob-like processes which extend into the under- lying mesenchyma. At the time they appear no muscularis mucosae is present. In embryos of 99 mm., 110 mm., 140 mm., the muscularis mucosae is still not visible; nevertheless, the intestinal glands all reach a certain depth, so that in a section, a line drawn parallel to the surface of the mucous membrane would practically touch the bottoms of all the glands. It is along this line, or rather, slightly below it, that the muscularis mucosae is becoming visible in an embryo of 187 mm. It is seen as a slight condensation of the mesenchyma forming a cir- cular band of about 0.014 mm. in thickness. In it are observa- ble, though not very distinctly, developing myoblasts. The band is slightly more distinct at 190 mm., and separates the connective tissue of the submucosa, which is condensed and well stained, from that of the tunica propria, which is loosely arranged and only faintly or not at all stained. At 187 and 190 mm. the basal ends of the glands can be seen resting upon this layer of muscle. Further formation of glands As the glands are gradually increasing in number as the growth of the embryo proceeds, the question arises, how are new glands formed? Do they develop like new villi, by evaginations of the epithelium between those already formed, or are they devel- oped after the manner which Patzelt has described, by a longi- tudinal splitting of those already present? In his discussion of this point Patzelt says: DEVELOPMENT OF THE LARGE INTESTINE 211 Noch eriibrigt es mir, die Art und Weise anzugeben, wie sich die Lieberkiihn’schen Driisen vermehren. Bei der Durchsicht der Prapa- rate, hauptsichlich aus den alteren Stadien, findet man oft im Grunde etwas verbreiterte Driisen. In der Mitte des verbreiterten Grundes erhebt sich ein Epithelhéckerchen. Oft auch ist dieses Hockerchen nicht mehr blos aus Epithelzellen gebildet, sondern gleicht im Durch- schnitte einem mit Epithel tiberkleideten Zé6ttchen, welches in das Innere der Driise hineinragt. Es entspricht dem Durchschnitte eines kleinen Fialtchens, welches den Grund der Driise in zwei Theile spaltet. Dieses Faltchen wichst immer hodher und hodher. Die Driise hat endlich das Aussehen, als ob in einem gemeinsamen Vorraum zwei Drii- senschliuche miindeten (Fig. 29 e). Wenn schliesslich die Héhe des Faltchens in gleicher Ebene mit der Innenfliche des Darmes steht, ist der Theilungsprocess vollendet, es sind aus einer Driise zwei geworden, He further believes that the upward growing connective tissue papilla continues its upward growth after it reaches the surface level and thus gives rise to a new villus. In further support of Patzelt’s view regarding gland multi- plication may be mentioned that if new glands appeared as new outward growths from the epithelium, then one would expect to find always in any fetal intestine glands which extended for varying distances toward the muscularis mucosae. How- ever, such an appearance is never found. The basal ends of the glands, as seen in figures 5, 6, 7, 8, 24 and 25, all extend down to one general level, no intermediate lengths being present. The branched types of glands are always present, some with two, some with three, and some with even four divisions. The amount of bifurcation also varies as Patzelt has figured. As in all problems of growing structures, it is indeed difficult to say precisely what changes are taking place, but I believe it safe to say that in this case that additional glands are developed by a longitudinal splitting of those already present. In the transverse colon of an embryo of 200 mm. practically the same conditions as those described for the sigmoid colon of the 190 mm. stage are encountered. The mucous membrane, although not thrown up into folds, does not appear stretched out. The villi are of various sizes, the tallest being from 0.14 to 0.18 mm. high. The intestinal glands measure from 0.18 to 0.22 mm. in length. Other measurements taken are as follows: THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO. 2 212 FRANKLIN PARADISE JOHNSON i a) Diametenot epithe lialiitilo Coe serine air ean enn 3.5 mm. Perimeter, measured at bases of glands.......................11.1 mm. Thicknessiof Mucosa eee Ee Lee Ee ato: Hani ine 0.40 mm. MhicknesstotOuverslyelsneeree Meee eer ern eet cee 0.27 mm. Number of glands touching muscularis mucosae..............75.0 Number of glands per running millimeter......................6.7 In the iliac colon of the same embryo, a slight distention is present as is indicated by the thickness of both the mucosa (0.32 mm.) and the outer layers (0.18 mm.). Although the preservation of the embryo is poor, enough can be made out from it to see that both villi and glands are shorter and broader than in the transverse colon. In an embryo of 240 mm. the transverse colon shows some distention, but not so much as was seen in this portion of the large intestine at 190 mm. ‘This is made evident by compar- ing the thicknesses of the mucosa and the outer coats. As seen in a model (not figured), made at double the magnification of the former model, the mucous membrane presents on its lumen surface a number of large irregular anastomosing folds. Be- tween the folds are clefts, which in some places appear to be the glands themselves; in other places they are merely crypts into whieh the glands open. The glands are, for the greater part, of the simple tubular type, but some show bifurcations into two or three portions. The epithelium, which varies in thickness from 0.020 to 0.034 mm., is made up of the typical simple columnar cells found in the adult intestine, and contains large numbers of goblet cells. The following list of measure- ments is given for comparison with the other stages: Noval diameter of epithelial tubes. ee eee os ee eno. Perimeter, measured at the bases of the glands............... 14.8 mm. MnicknessrOLmMMlCOSa; ccc schon. Miata emir ensue okie nae 7 0-36 Thicknesstol OuleriCOatsen. a acs ceeetaene pretewe mere antics ier ete 0.22 mm. Number of glands touching muscularis mucosae.............. 114.0 Number of glands per running millimeter .................... 7.7 In an embryo of 320 mm. a piece of transverse colon which was greatly distended with meconium was examined. The large folds of the mucosa have all disappeared and the intestinal wall DEVELOPMENT OF THE LARGE INTESTINE AAS as a whole is considerably thinned out. Owing to poor preser- vation, however, it was not possible to determine the presence of villi and folds. Measurements made are as follows: Total diameter of epithelial tube....................:.. D2 Onxe 1425) mim: Perimeter measunedmasibekOnen. 45 4.425. 4+ 40056 08 seen 345 moma: IM ane] MESS Cie IMMDNKOEC, sodwad oa abase oon donneeon oboe oe eee 0.16 mm. IIMOSACES Oil CUMS COB 6oceo.6 600d ooncubsbacuonesscosscueut 0.20 mm. INumobersofeclanc sect tten cas cree ener ince ray use eee ard oe vc oe 269.0 Numbertoticlands per millimeter sncknco tt: oes: ee. eo ss ena oe Gol In a fetus of about seven months (premature birth) the ascend- ing colon seems to be almost entirely devoid of villi and the before-described folds. Although not modelled, it is plain to see that the surface epithelium is for the most part, though not entirely, level. At more or less regular intervals, it dips down into the eylindrical intestinal glands. Measurements: (otal diameter of epithelial tube. .:..-..6--4..5...40+5.0--2. 98-7 “mm. TEU CGE Tose nee see once cee ahescad See a | een Bl Siete MN 18.5 mm. fh CKMesss Olam COSAA. sme oe cesta on ee eee ene eee 0.27 mm. ‘TnI Oh QUEL COIS inavocneaeeenecdducasodceseseecuacée 0.54 mm. iINwuimoberroteclandsintirs mrss sae Soca eee rae eee 162.0 Number of glands: per millimeter....5..) 5... s006. ot eaees: 8.7 Sections of the sigmoid colon show a similar condition as regards glands. They are of an equal length and similarly dis- tributed. Fig. 8 Cross section of the mucosa of the transverse colon of a human embryo ait lomo, »< (0), In a fully developed fetus at birth (fig. 8), a condition is reached in which the villi have entirely disappeared. A model of a por- tion of the epithelium of the transverse colon is shown in figure 25. As seen from the model, only glands are present, the open- ings of which appear irregular when viewed from the lumen 214 FRANKLIN PARADISE JOHNSON side. For the most part the mouths of the glands open singly to the surface, but in one region of the model, three can be seen opening together in a slightly depressed region of the surface epithehum. The glands are still of the simple and bifurcated types, the bifurcated ones representing different stages in the process of splitting. The epithelium, which is similar to that in the adult, is 0.017 mm. in thickness at the surface, while in the glands it measures 0.28 mm. Goblet cells are still numer- ous. Measurements made from cross sections of the transverse colon at birth are as follows: mm. Diametemotcepiunelialmculoe=ter ee renee eee eee re 5.5 CRIM CCC ref ocr ors MA en erierd od ala ote ache 39.0 mm. cbnekiesso tema COSA sere .,; oe > hae eee ee ee ee 0.25 mm. MIGNON Ou CUM COM oacadcaescncnecacevopesoddsaedocy 0.27-0.72 mm. Niamb ere igor amd State ysiabars oc, aed eather cee er nee 384.0 Numberof lands per muillimetensy..211. 494) eee a) Wists) Summary To the foregoing observations the following summary may be added. As has been pointed out before, the cylindrical tube of epithelium of the early stages develops longitudinal thickenings or ridges. In an embryo of 58 mm. these are becoming par- tially transformed into low longitudinal folds. In embryos of 65 mm. these folds apparently segment, and in embryos of 88 to 99 mm. true villi are present. We have, therefore, longi- tudinal folds apparently subdivided to form villi. This view concerning the formation of the villi in the small intestine has been presented by Berry (’00) and confirmed by Forssner (’07). In epposition to this, the view that the villi arise as separate growths of the epithelium, not preceded by folds, has been maintained by Koelliker (61 and ’79), Barth (68), and Brand (77). Voigt (99) believed that the surface epithelium was cut up into a number of elevations by a net work of fissures and furrows, and that these elevations grew into vill. In a study of the development of the whole of the human small intestine the present author (10) wrote: DEVELOPMENT OF THE LARGE INTESTINE Zhe In briefly summarizing the development of villi, it may be said that the general tendency throughout the whole of the small intestine is for villi to develop as separate invaginations of the epithelium. Owing however, to the occurrence of transitory structures (vacuoles, diverti- cula, and folds) their development is manifested differently in differ- ent parts of the intestine. Although in the large intestine formation of villi is preceded by distinct longitudinal folds, it does not seem probable to me, after a study of my models, that there is a mechanical segmen- tation of the longitudinal folds. It must be remembered that the epithelial tube is ever growing by an increase in the number of its cells, and by the enlargement and duplication of its parts. Because of this active growth one would expect, therefore, villi to form by an active direct process, rather than by an indirect one. It seems more probable to me, that small knob-like eleva- tions are developed along the tops of the folds, and that these knobs form into villi. If such were the case, the picture pre- sented would always be one which would appear like a segmen- tation of folds. As the villi grew taller and taller, the segment- ing fissures would appear to be sinking in deeper and deeper. It is important in this connection to note that the original folds are considerably smaller than the villi, a fact which is in favor of the view just proposed. Regarding the further development of villi, it may be said with certainty, that they arise separately in between those already present. The earliest glands develop as small knob-like growths of the epithelium into the mesenchyma. As has been pointed out before, the additional glands are probably formed by a longi- tudinal splitting of those already present. The villi reach their maximum height in embryos of 110 mm. to 140 mm. in length. From this stage on they gradually be- come smaller and smaller. OO So _ 10) Contracted | 40 cm. below valve | Distended 55 em. 5.8 | 0.45 | 0.07 0.36418 Ose |)" 40709, 1 006 | below | valve 1 Not including muscularis mucosae 2 Average 3 Including muscularis mucosae 4 Highest 5 Deepest in the contracted. In table 3 measurements from both the normally contracted and the normally distended intestines are given. The large intestine in the filled condition also shows a marked shortening of its glands, as can be seen by comparing figures 8 and 9. Artificial distention Some of the results of artificial distention produced by pres- sure are given in table 4. As would be expected, different amounts of distention produce different degrees of shortening 242 FRANKLIN PARADISE JOHNSON TABLE 4 Measurements showing the effects of normal distention of the large intestine as con- trasted to those of contracted CONDITION OF | pagron | pyaar, | Se SEANDS Loe INTESTINE | (mean) Depth! | Breadth? | CUTER COATS? mm. mm. | mm. mm, Contracted 30 cm. BAU 0.27 | 0.045 0.32 below valve | | Distended 45 cm. 6.4 0.13 0.062 0.06 below | valve } 1Tn this case the thickness of the glands is equal to the thickness of the mu- cosa; muscularis mucosae omitted; depth longest. 2 Average. of the glands and villi. In figure 2 is shown a section of the small intestine of a newborn guinea-pig distended by means of a column of water 150 cm. high. The glands throughout the whole piece of intestine have practically disappeared. In an adult guinea-pig, however, stronger distention (pressure about 270 em. water) did not do away with glands to so great an extent although in some places they are entirely gone. Those few which remain are short and very broad. Distention of the large intestine (pressure of 150 c.m. of water) reduces the length of the glands considerably. They were further shortened by a pressure of about 270 cm. of water, but did not entirely dis- appear. The epithelium also shows the effects of strongly distending the small and large intestines. Whereas in the contracted intestine the epithelium is composed ‘of tall cylindrical cells, with rounded or elongated nuclei, in the strongly distended intestine it is much flatter, and is composed of cuboidal cells” with nuclei which are also flattened. In figures 10 and 11 are shown the effects of strong distention of the large intes- tine upon its epithelium. Similar pictures may be obtained from the small intestine. —— EFFECTS OF DISTENTION OF THE INTESTINE 243 Contraction following artificial distention A piece of small intestine was distended at a pressure of 150 em. of water with warm normal salt solution. The pressure was maintained for about one minute and was then removed and placed immediately in Zenker’s fluid. Contraction began in a few seconds time. In figure 3 is seen an empty piece of intestine taken from the region adjacent to the above. The distended and subsequently contracted intestine is shown in figure 4. Figure 7 shows the effects of a pressure of 150 cm. of water upon an adjacent piece of small intestine. Similar results were obtained with the large intestine. THE INTESTINES OF OTHER ANIMALS Cat. Cross sections of the empty small intestine of the cat fixed in Zenker’s fluid show five to six low longitudinal folds of the mucous membrane. On these folds both the villi and the glands are longer and broader than those in the spaces between them, suggesting that the folds are regions of more strongly contracted mucosa. In the normally expanded intestine, how- ever, the villi and glands are everywhere more uniform in size. The effects of distention, both normal and with a pressure of 150 cm. of water are shown in table 5. TABLE 5 Measurements showing the effects of distention of the small intestine of the cat upon the shape of villi and glands DIAMETER Soe GLANDS CONDITION OF INTESTINE OF EP. | cS TUBE Height Breadth | Depth Breadth mm, mm, mm. mm. mm. Normally contracted............ 5.8 1.06 0.13 0.80 0.045 Normally distended............. 8.0 0.74 0.16 0.42 0.060 Experimentally distended.......| 10.0 0.69 0.18 0.37 0.061 In the large intestine of the cat the glands are likewise short- ened and broadened during distention as shown in table 6. Dog. Artificial distention on the dog’s intestine under a pressure of 7 pounds per square inch (bursting point, 9 pounds) 244 FRANKLIN PARADISE JOHNSON TABLE 6 Measurements showing the effects of distention of the large intestine of the cat wpon the shape of glands tae Pe | GLANDS | DIAMETER OF CONDITION OF INTESTINE =a HE yee Depth Breadth F mm. mm. “mm. Normally contracted............ | 135 0.45 | 0.056 Normally distended.............| 15.0 0.27 | 0.070 did not result in a complete disappearance of either glands or villi. The effects were largely similar to those on the villi and glands of the cat. ‘mf TABLE 7 Measurements showing the effects of distention upon the small intestine of the dog upon the shape of villi and glands VILLI GLANDS CONDITION OF INTESTINE Height Breadth Depth Breadth mm. mm. mm. mm. Gomtraiched awe. see eee 1.26 0.22 0.81 0.03 SDIShENGedlanstian aie ae 0.40 0.32 0.13 0.07 Mouse. Experimental distention of the mouse’s small intes- tine at a pressure of 100 cm. of water caused a greater shorten- ing of glands than of villi. The appearance obtained is some- what similar to that produced by distending the intestine of the guinea-pig, except that the villi of the mouse are relatively taller and are not folded on themselves. Table 8 shows the amount of shortening produced. TABLE 8 Measurements showing the effects of distention of the small intestine of the mouse upon the shape of villi and glands VILLI GLANDS CONDITION OF INTESTINE rs Height Breadth Depth Breadth mm, mm. mm. mm, Contracteulenehrontes suse 0.18 0.09 0.11 0.04 Distendedy) arty. ise. 50 au Qald ys 20-8 0.04 0.07 EFFECTS OF DISTENTION OF THE INTESTINE 245 Distention of the stomach and oesophagus Before concluding the present work it seems desirable to call attention to the effects of distention upon the mucous mem- branes of the stomach and oesophagus. Strong distention of the stomach of the cat and of the guinea-pig brings about a thinning out of the mucosa, a shortening of both pits and glands which at the same time are widened and spread apart. In the oesophagus of these animals strong distention brings about a marked flattening of the stratified squamous epithelium and an apparent reduction in the number of its cell layers. Thus, the oesophageal epithelium of the cat which is normally com- posed of 13 to 18 layers of polygonal cells, on strong disten- tion appears to consist of 6 to 8 layers of very much flattened cells. The effects here upon the epithelium of the oesophagus are somewhat comparable to those which may be produced upon the epithelium of the ureter through distention as described by Harvey (’09). CONCLUSIONS The effects of distention of the intestine may be enumerated as follows: 1. The outer intestinal coats become reduced in thickness. 2. The mucosa becomes reduced in thickness. 3. The villi become shorter and broader. 4. Glands become shorter and broader. In the guinea-pig and mouse they may entirely disappear if the intestine is strongly distended. | 5. In the intestine of the guinea-pig the epithelium becomes flattened upon strong distention. It is evident from the foregoing results that the shapes of villi and glands are to a great extent dependent upon the con- dition of distention or contraction of the intestine. This is true not only for marked distention produced experimentally, but for the smaller amounts of distention which take place under normal conditions. It seems probable, therefore, that with each dilation and contraction of normal peristalsis and 246 FRANKLIN PARADISE JOHNSON of the rhythmical movements of the intestine, the villi change their shape, and in this way bring about a more thorough mix- ing of the intestinal contents. Moreover, by the unfolding of the intestinal glands, a greater amount of epithelium is ex- posed to the intestinal content, and thus the absorption sur- face is increased. Because of the variety of shapes presented by the gland cavities it is not possible, by ordinary methods, to determine their exact capacities, but it is probable, that the capacities of the glands are decreased upon strong distention, and their contents are then partially emptied into the lumen of the intestine. BIBLIOGRAPHY Brtcxe, E. 1850 Ueber den Bau und die physiologische Bedeutung der Peyeri- schen Driisen. Denkschr. d. Akad. zu Wien, Bd. 2, pp. 21-26. Busarp, E. 1909 Etude des types appendiciels de la muqueuse intestinale, en rapport avec des régimes alimentaires. Inter. Monatschr. f. Anat. u. Phys., Bd. 26, pp. 1-96. GruBy, AND Deruaronp, O. 1843 Résultats des recherches faites sur l’ana- tomie et les fonctions des villosités intestinales, l’absorption, la pré- paration et la composition organique du chyle dans les animaux. Comp. rendus, Acad. Sci. Paris, tom. 16, pp. 1194-1197. Harvey, R. W. 1908 Variations in the wall of the large intestine and in the number and staining properties of goblet cells. Anat. Rec., vol. 2, pp. 129-142. 1909 Variations with distention in the wall and epithelium of the bladder and ureter. Anat. Rec., vol. 3, pp. 296-307. HeitzMANN, C. 1868 Zur Kenntniss der Diindarmzotten. Sitz. d. k. Akad. Wien, Bd. 58, Abth. 2, S. 253-268. 1883 Microscopical morphology of the human body. New York. K6uurKerR, A. 1851 Ueber das Vorkommen von glatten Muskelfasern in Schleim- haiuten. Zeitschr. f. Zool., Bd. 3, pp. 106-107. LaucacuHig, A. 1843 Mémoire sur la structure et le mode d’action des villo- sités intestinales. Comp. rendus, Acad. Sci. Paris, tom. 16, pp. 1125- 1127. Matt, F. P. 1896 A study of intestinal contraction. Johns Hopkins Hospital Reports, vol. 1, pp. 37-75. Verson, E. 1871 Diinndarm, in Stricker’s Handbuch der Lehre von den Gewe- ben. Leipzig. PLATE 1 EXPLANATION OF FIGURES Normally contracted small intestine of a newborn guinea-pig. X 80. Small intestine of newborn guinea-pig distended with 150 cm. water pres- sure. X 80. 3 Normally contracted small intestine of adult guinea-pig. > S80. 4 Small intestine of same guinea-pig subsequently contracted after disten- tion similar to that in figure 7. X 80. PLATE 2 EXPLANATION OF FIGURES 5 Strongly contracted small intestine of adult guinea-pig. X 80. 6 Small intestine of adult guinea-pig normally distended with food mate- rial. X 80. 7 Small intestine of adult guinea-pig distended with 150 cm. of water pres- sure. X 80. PLATE 1 EFFECTS OF DISTENTION OF THE INTESTINE FRANKLIN PARADISE JOHNSON EFFECTS OF DISTENTION OF THE INTESTINE PLATE 2 FRANKLIN PARADISE JOHNSON PLATE 3 EFFECTS OF DISTENTION OF THE INTESTINE FRANKLIN PARADISE JOHNSON Fig. 9 Fig. 10 Fig. 11 EXPLANATION OF FIGURES 8 Normally contracted large intestine of adult guinea-pig. 80. 9 Normally distended large intestine of adult guinea-pig; (distended with gas). > 80. 10 Surface epithelium of normally contracted large intestine of adult guinea pig. X 720. 11 Surface epithelium of greatly distended large intestine of adult guinea- pig. X 720. 250, HISTOLOGY OF THE SENSORY GANGLIA OF BIRDS! E. VICTOR SMITH FORTY FIGURES CONTENTS NATE O MIC CLOT see rrer thee, ssi encre ene ers 2 Get CRORE UME BL oN a 252 PeeSricl comments on the-literatures. 22 3345 so.0e dsc bees oo eck cited. 252 PUPA ee LONI Ser so) se ao teres TEL PRE ry Pn Tad wee Oe 255 ee SeMSOnueaneliazOrghe Chick... 4 a) /ae Seton Se a 256 *) SS y OVE SINE a i ae ees o NEs s 257 Sey (OVS 3) 2M 2475 1 bE eR ie 258 aGunglon of the ninthnerveusss sae. tee oes ole ose oe 258 barlheavarus: Pan glion. “cree weet eee ree ty 259 Gay iceiG aeserian ganglion. somerebee ececlic he le be oa oe 260 Bereusotyrraneia Of the Owl... wih. sos coe eis weet eee ees 264 Un GelsTiNe Ter) RRA Sete goed «| doin, ok Oke ee eS 264 Be ete prerie Pemba 2) ee occt. cs Reet ea ene a ity STA Te 265 ee DERVA PUSS PAN PION : 707.8 /.e ot seen ee eet Uke ae Mate we) 0) 265 [oe eV ASSCLIAN SANCTION es te ota eA eee Ser oe tee Ao 266 er Ganglion of the auditory merve??..0 3)5)0.0..25-9-.0.e 266 Owpensoryeaneliacor the goo0se:.) 2054 |e ee eae. 267 VASO MUN) Sa arene Sie Os mk at SOA Na a mt te ae 267 PSOE 01 Poh aol he rr Se ee NE AIR ee eee eye a UE bes Dae 268 aeGangion or the ninth nervel: ss vases cos Monee 268 bredibe varus ganglion): -. van! 800. 2,3, 4,5 Bipolar cells from spinal ganglia of the adult chick. Figure 5 shows the protoplasmic network of the cell body and of the processes. Note in the differ- ent cells the capsule, capsular nuclei, and the smaller size of the central process. xX 800. 6 Bipolar cell in which two processes have just come together. Observed in a spinal ganglion of an adult chick. X 800. 284 HISTOLOGY OF SENSORY GANGLIA OF BIRDS PLATE 1 E. VICTOR SMITH C.pr, Cap.nu. 5 285 PLATE 2 EXPLANATION OF FIGURES 7 Cell from the spinal ganglion of the chick, the main process having an initial glomerulus. > 800. 8 A spinal ganglion cell of the chick having fenestrations and slings near the periphery. X 800. 9 Cell of the vagus ganglion of a six-year-old hen showing fenestrations, slings, and pericellular network. > 800. 10 Cell from the same ganglion as figure 9, showing fibers surrounding the cell, and also part of the pericellular network of an adjacent cell. > 800. 11 Much convoluted axis cylinder from the Gasserian ganglion of the chick, | stained with silver nitrate and counterstained with Delafield’s haematoxylin; the ends a and b should be continuous. X 2000. PLATE 2 HISTOLOGY OF SENSORY GANGLIA OF BIRDS E. VICTOR SMITH slo. fen. NT TIT 287 PLATE 3 EXPLANATION OF FIGURES 12 A section through the central part of the glossopharyngeal ganglion of the chick, indicating the relations of the elements. 13 A group of cells from the periphery of the vagus ganglion of the chick, showing typical cells and bundles of non-medullated fibers. X 1000. 288 HISTOLOGY OF SENSORY GANGLIA OF BIRDS PLATE 3 E. VICTOR SMITH PLATE 4 EXPLANATION OF FIGURES 14 Cell from the vagus ganglion of a six-year-old hen showing pericellular net- work. X 800. 15 Cell with initial glomerulus from the Gasserian ganglion of the chick. X 800. 16 Cell with twisted process from the Gasserian ganglion of the chick. X 800. 17 Vacuolated cell from the Gasserian ganglion of the old hen. S800. 18 Multipolar cell with accessory processes and protoplasmic slings, from a sympathetic ganglion of the chick. X 800. 19 Cell showing accessory processes terminating in end bulbs, from the Gas- serian of a six-year-old hen. X 800. 290 PLATE 4 HISTOLOGY OF SENSORY GANGLIA OF BIRDS SMITH VICTOR E. 291 PLATE 5 EXPLANATION OF FIGURES 20 Bipolar cell with fenestration and accessory process near the central process, from the Gasserian of a chick. X 800. 21 Cell from the Gasserian ganglion of an old hen, showing slings about the axone and also at the opposite pole. 800. 22 Cell from the Gasserian ganglion of an old hen with slings and protoplasmic strands about the axone. X 800. 23 Cell with a nest of protoplasmic loops on the pole opposite the main process, from the Gasserian ganglion of a six-year-old hen. X 800. 24 Bipolar cell with protoplasmic loops and an accessory process near the cen- tral process, from the Gasserian ganglion of a six-months-old chick. 800. 292 HISTOLOGY OF SENSORY GANGLIA OF BIRDS PLATE 5 E. VICTOR SMITH fen. ac.pr. 293 PLATE 6 EXPLANATION OF FIGURES 25 Lobed cell with glomerulus and accessory processes, from the vagus gan- glion of the owl. X 800. 26 Irregular cell from the vagus ganglion of the owl. 800. 27 Cell from the vagus ganglion of the owl. X 800. 28 and 29 Cells from the vagus ganglion of the owl showing fine accessory processes with end bulbs issuing from the main process. X 800. 30 Cells from the vagus ganglion of the owl showing twisted process with fine accessory processes emerging from the main process. XX 800. 31 Lobulated cell from the Gasserian ganglion of the owl, with fenestrations and accessory processes. > 800. 32 A lobed cell with a vacuole from the Gasserian ganglion of the owl. > 800. 33 Cellfrom the same ganglion showing fibrillar network and initial glomerulus. X 800. 34 Bipolar cells from the ganglion of the eighth nerve of the owl. 800. 294 HISTOLOGY OF SENSORY GANGLIA OF BIRDS PLATE 6 E. VICTOR SMITH -. p.pr PLATE 7 EXPLANATION OF FIGURES 35 Bipolar cell from the ganglion of the eighth nerve of the owl showing a depar- ture from the oppositi-polar condition. X 800. 36 Cell from the auditory ganglion of the owl, showing glomerulus on the peripherally directed process. > 800. 37 Typical cell from the glossopharyngeal ganglion of the goose. X 800. 38 800. 40 A group of cells from a dorsal spinal ganglion of the sparrow. 296 PLATE 7 HISTOLOGY OF SENSORY GANGLIA OF BIRDS VICTOR SMITH E. 297 AT ILO UL DULLAVIG 1ymipuoia UIsSsUue ITN tne 1rog, the ODS ervations FURTHER STUDIES OF THE HISTOLOGY OF THE THYMUS ALWIN M. PAPPENHEIMER From the Marine Biological Laboratory, Woods Hole, Mass., and the Department of Pathology, College of Physicians and Sinpeane. Coinantaa University, New York TEN FIGURES (FIVE PLATES) mh SS . 1p ey See ea = = ERRATA The American Journal of Anatomy, Volume 13, Number 4, September, 1912 Page 428, description of figure 8, for Ceriarcuon fig. 32) read (Reconstruction, fig. 29). Page 438, third line, description of ae 13, for figure 33 read figure 30. Page 442, figure 16, for 4.5 read 4.5. Page 472, description of figure 32, for figure 31 read figure 28. Page 473, figure 33, for 5d and 5s, read 4d and 4s. were made on tissue obtained from young rats. It may be admitted at the outset that clear-cut morphologi- eal evidence of a secretory function on the part of any of the thymic elements, has not been obtained. The observations which were made serve, however, to clarify some of the contro- _versial points in the normal structure of the organ and add some new histological details which seem to justify their publication. 4 299 | THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, No. 3 MARCH, 1913 FURTHER STUDIES OF THE HISTOLOGY OF THE THYMUS ALWIN M. PAPPENHEIMER From the Marine Biological Laboratory, Woods Hole, Mass., and the Department of Pathology, College of Physicians and Surgeons, Columbia University, New York TEN FIGURES (FIVE PLATES) The conception 'of the thymus gland as an organ of internal secretion rests almost wholly upon the facts obtained from physi- ological experiment. In the thyroid, parathyroid, hypophysis and adrenal, we have clear morphological evidence of secretory activity on the part of the parenchymal cells. But this is not true in the case of the thymus, and we do not even know which of the complex elements of the gland contribute the hypothetical secretion. The following study of the frog’s thymus was under- taken primarily in the hope of throwing some light upon this problem, by the use of methods which have not hitherto been applied to a study of the thymus; namely, stains for the demon- stration of cell granulae, and the study of the living cells grown in vitro after the method elaborated by Harrison, Burrows and Carrel. The findings in the fixed tissue were checked up and amplified by applying various vital stains to the living cells in cultures. The work includes also a comparative study of the erowth of thymus and lymph-node in vitro. Because of the absence of suitable lymphoid tissue in the frog, the observations were made on tissue obtained from young rats. It may be admitted at the outset that clear-cut morphologi- cal evidence of a secretory function on the part of any of the thymic elements, has not been obtained. The observations which were made serve, however, to clarify some of the contro- versial points in the normal structure of the organ and add some new histological details which seem to justify their publication. 299 THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, No. 3 MARCH, 1913 300 ALWIN M. PAPPENHEIMER MATERIAL AND TECHNIC The glands were obtained during the months of July, August and the early part of September from freshly caught specimens of Rana clamata; a few also from spotted frogs shipped from a distance and showing in consequence of the prolonged starvation, marked involutional changes. In addition to the usual histo- logical methods a variety of granule stains were used: Altmann’s acid fuchsin, as originally described and as modified by Lane; Bensley’s neutral gentian violet (1); Heidenhain’s iron-alum hematoxylin after fixation in Benda’s modified Fleming’s solu- tion; and Benda’s mitochondrial stain, used in the manner origi- nally described by him and according to the modification sug- gested by Meves and Duesberg (2). The clearest pictures were obtained with the modified Benda method, although somewhat varying appearances were often presented by sections prepared with the same technic. The preparation of in vitro cultures has been so often described that it is unnecessary to give the method again in detail. The plasma was obtained directly from the frog’s heart by aspiration with an oiled needle or glass pipette. Hanging drop cultures were kept at room temperature, as it was found that all the elements rapidly degenerated when incubated at 37°. In study- ing the reaction of the cells to various vital stains, dilute sterile solutions-of the dye in Ringer’s fluid were added directly to the cultures. In this way, with the gradual diffusion of the dye through the plasma, the reaction of individual cells could be directly observed. In other cases, the vital stain was added to the plasma before implanting the tissue, but under these condi- tions, for reasons which will be discussed later, no growth was observed. HISTOLOGICAL STUDY In the adult frog, the thymus is an elliptical, slightly flattened yellowish body, from 2 to 4 mm. in length, situated on either side beneath the angle of the inferior maxillary, and in close relation to the glosso-pharyngeal nerve and the depressor man- HISTOLOGY OF THE THYMUS 301 dibulae muscle. On the surface, one may see in the gross stellate masses of black pigment. The surface is smooth and shows no lobulation. After prolonged starvation, or in infected frogs, the gland shrinks, has a somewhat translucent appearance and may be difficult to recognize with the unaided eye. In old frogs, the texture of the gland becomes firmer and the color a deeper yellow. Microscopically, one finds a sharp distinction between cortex and medulla, the relative proportions varying in different indi- viduals. The cortex is composed principally of closely packed small thymus cells, but often shows a subdivision into an outer zone, in which the nuclei of the small cells are paler and show a more distinct chromatin network; and an inner zone, in which the nuclei are smaller, denser and more deeply stained. Mitoses in variable number are present in both strata of the cortex, but are probably more abundant in the periphery. The medulla is formed in large part by the myoid cells, which in the frog’s thymus are often a striking and conspicuous feature of the histological picture. These cells have in general an oval or circular outline which is quite sharply defined, although band-like forms occur. With Heidenhain’s iron hematoxylin or with crystal violet after Benda fixation, they show a distinct fibrillar structure. The fibrils most commonly have a circular arrangement, and show an alignment of deeply staining rods or granulae, giving the appearance of distinct cross striation. In many cells of this type, however, there is no regular alignment of rods or granules, which may be quite irregularly disposed throughout the cell. Whether the granulae represent cross sec- tions of short filaments, or true granulae, is often quite difficult to decide. Many cells show in their peripheral portion, regularly disposed striated fibrils, while in the central portion of the cell, these appear to be broken up into dots and lines. Cells of this type containing scattered clumps of deeply stained material, are interpreted as degeneration forms. When the granules or rods are at the same level in adjacent fibrils, the resemblance to the striations of muscle fibers is a close one, and the aptness of the term myoid is evident. 302 ALWIN M. PAPPENHEIMER Very frequently, these cells contain one or more rounded cav- ities, and in some eases the cell is composed merely of a shell of striated fibrils, enclosing a single. large central cavity. In one such cell observed in a living culture, the cell, which in its free condition had taken on a globular form appeared to be tunnelled by a cavity the rim of which was slightly more refrac- tile and presented a distinct striation. The nucleus was appar- ently located in a lobular projection at one portion of the cell. Two views are held as to the nature of the myoid cells,—one supported by Mayer (3), Pensa (4), Weissenberg (5), and others, that they are derived from muscle fibers which become included in the gland in the course of its development; the other sup- ported by Hammar (6) and Pappenheimer (7), that they are modified elements derived from the reticulum. In sections, the appearances lend countenance to the latter view: fibrils may occasionally be traced from one cell to another and transitions between reticular cells and cells with poorly developed fibrils apparently occur. In teased preparations, these elements may be readily recognized by their large size, regular contours, refractile appearance, and by the presence of striations some- times seen on careful focussing. They appear always under these circumstances, as sharply cireumscribed, usually globular bodies, so that we must assume that their anatomical connec- tion with the reticular syncytium, is extremely slight. Their capacity for migration and growth in plasma cultures, is also nil. They show no amoeboid activity, but retain their fixed form, accumulate fat and gradually degenerate, in marked con- trast, as will be pointed out later, to the reticular elements. Evidence of contractility on the part of the fibrils was carefully looked for, but an abrupt change of form suggesting such a function, was not observed. In sections, the reticular cells are differentiated from the small thymic cells principally by the character of their nuclei, which are larger, paler with a more distinct chromatin network. The outlines of these cells are indistinct, the cells forming a loose protoplasmic meshwork in the interstices of which lie the myoid cells and the small thymic elements. In the cortex, amongst ss HISTOLOGY OF THE THYMUS 303 the closely packed small cells, one finds scattered nuclei of the large type, evidently belonging to the reticular cells, but the protoplasmic reticulum is obscured. In the ordinary hematoxylin-eosin preparations, or in sections stained with polychrome methylene-blue eosin, there are regu- larly seen scattered cells with large, rounded eosinophilic granu- lations. These cells are found both in the cortex and the me- dulla, and are often but not always, in close relation with the connective tissue sheaths of the blood vessels. I have been unable to decide from my preparations whether: these granulae always belong to leucocytes or myelocytes, or whether some of the reticular cells may not contain eosinophilic granules. The foregoing brief description, which agrees in all essentials with that of previous workers on the amphibian thymus, but which ignores entirely the mooted points as to the histogenesis of the different cellular components, will however suffice for the present study. I wish now to describe the appearances found in sections fixed and stained according to Benda’s method for the demonstration of mitochondria. In successful slides, the nuclear chromatin does not retain the erystal violet, but is stained yellowish brown by the alizarine. When, for reasons which were not determined, the nuclei retain the violet stain, the cytoplasmic granules are much less readily demonstrable. These preparations, however, give excellent pic- tures of the mitotic figures, of the fibrils of the myoid cells, and bring out also certain coarser granules in the reticular cells, to which we shall refer later. In good preparations, the nuclei, though unstained with the violet, are distinct. The chromatin of the small cells is in the form of clumps, from one to four in a nucleus, lying in an un- stained clear space. Ragged threads extend from these to the nuclear membrane. The latter shows a tendency to retain the crystal violet, especially when differentiation is not carried too far. The nuclei are thus often bounded by a rather heavy purplish line. It is not possible to distinguish absolutely between the nuclei of the small cells and those of the larger reticular elements. In general, the latter show a more delicate and evenly 304 ALWIN M. PAPPENHEIMER distributed chromatin network, which is not in the form of large discrete clumps. The cell limits cannot le made out clearly, even in very thin sections (24). Occasionally in the cortical portion, the small thymic cells show a distinct outline, the nu- cleus being bounded by a narrow rim of brownish protoplasm, which is thicker at one pole of the cell, where the nucleus com- monly shows a slight dell or indentation. In the medulla, where the different types of cells are intimately commingled, the nuclei appear separated by an indefinite protoplasmic substance. It is thus exceedingly difficult to determine to which cell the cytoplasmic granulae belong. Scattered between the nuclei, but never in or upon them, are very numerous minute purple granulae (fig. 1). These vary somewhat in size, but are in general smaller than the smallest coccus, and in some cells are barely within the range of visibility. (Comp. OC/6, Imm. 1/12 in.) The abundance of the granulae depends largely upon the degree of differentiation in acetic acid and alcohol. In sections which have been well differentiated, the granulae are fewer in number, but are more distinct, standing out sharply from the pale brownish background. Instructive pictures are found near the ragged edge of sections in areas in which the small thymic cells have been dislodged or fallen out, and only the protoplasmic reticulum persists. Here the protoplasmic meshwork is found studded with innumerable, minute sharply defined granulae. Whether the small thymic cells also contain granulae, or whether these are limited to the reticular cells, is difficult to decide from a study of the sections alone. The granulae are present in the cortical portion of the gland, in some sections in considerable abundance. Often they are in close relation to the nuclei of the small thymic cells, and in favorable cases, where the cells have become separated by a break in the section, they appear to lie within the narrow zone of protoplasm on one side of the nucleus. Often they form a row lying between adjacent cells, and they adhere to, or are incorporated with individual cells which have become loosened from their surroundings. From a study of the sections, the impression was gained that the small cells as well as the large reticular epithelial elements, contain HISTOLOGY OF THE THYMUS 305 granules (fig. 2), and this was confirmed by a study of the living cells by means of vital stains. While the protoplasm of the reticular cells has a fine fibrillary structure, it was not possible to demonstrate fila to which the granulae bore a definite arrangement. An. alignment of the granules in rows was not found in the reticular cells, in the sec- tions. In the myoid cells, however, the granulae or rods are arranged in definite rows at regular intervals upon a fibrillar groundwork, and to this is due the apparent cross-striation of the fibrillae and their resemblance to muscle fibers. While the granules and filaments of the myoid cells are readily demonstrable by all the mitochondrial stains, they appear with equal distinctness after formalin fixation and prolonged staining with Heidenhain’s iron hematoxylin after mordanting in 4 per cent alum. They are thus chemically distinct from the granulae of the reticular and small thymic cells which cannot be brought to view by this simple method. In sections stained according to the Meves-Deusberg modifi- cation of the Benda method, the granulae are less numerous but ’ more distinct. They occur in groups in certain of the reticular cells, and are quite variable in size, the largest being about half as large as the nucleus of a small cell; the largest granules or droplets may show partial decolorization. They are almost always spherical, but occasionally are slightly elongated and somewhat irregular (figs. 3 and 4). There remain to be noted certain cells with coarse slightly rod-shaped or bacillus-like granules, which stain intensely with crystal violet, even after fixation in Zenker-formol. These cells are scattered irregularly through cortex and medulla. They” are not numerous but by reason of their deep staining and the. large size of their granules, are very conspicuous. These cells are probably identical with the gentianophilic cells described by Prenant (8). Their identity with the eosinophile cells is ques- tionable, although Prenant suggests this possibility, and states that the eosinophiles of the blood are also gentianophilic. The elongated character of the granules would, however, serve to differentiate these cells from the eosinophiles in which the gran- ules are spherical. 306 ALWIN M. PAPPENHEIMER OBSERVATIONS UPON LIVING CELLS In the study of the living cells in vitro, certain difficulties were encountered, and the chief of these was the uncertainty in regard to the derivation of the growing elements. It was only after the study and comparison of a large number of different preparations that certain types of cells could be recognized, classified and their origin made reasonably certain. As has been observed by all workers, the growing cells do not adhere to their differentiated form, but assume a simpler and often indifferent type. The recent work of Foot (9) upon the growth of bone- marrow in vitro, emphasizes the difficulty in identifying the growing cells with definite constituents of the normal tissue. Even under approximately identical conditions, so far as they can be analyzed, there is considerable variation in the extent of the growth, and to a less degree, in its character. Thus of a given series, only a certain proportion yields a definite growth, others gradually degenerating. While such discrepancies are undoubtedly due in large part to technical faults, it is rarely possible to find the exact cause for the failure of growth. That the age of the frog from which the thymus is obtained has some influence upon the growth, is suggested by the follow- ing series. Two parallel sets of cultures were made, from half of which the thymus was taken from a young frog (6 em.), and for the others, from a very large frog (9 em.). The plasma of the small frog was used for both series. Eighteen preparations were made. Abundant growth was obtained in all but two of the cultures of ‘young’ thymus, whereas only three of the cul- tures of ‘old’ thymus grew and these but sparingly (33 per cent). In another series, eight preparations of thymus from an old very large frog, failed to show growth, while three controls of young frog thymus in the same plasma, showed abundant growth. There was unfortunately no opportunity to repeat these obser- vations on a large scale, but the point deserves further study because of its bearing upon the involution of the thymus in adult life. It might be supposed that the plasma of old animals would exert a deleterious influence upon the growth of the thymic elements, but this has proven not to be the case. Some of the ——— HISTOLOGY OF THE THYMUS 307 best growths obtained took place in the plasma of very large, and presumably old frogs. The influence of mechanical factors has been repeatedly em- phasized by workers with this method. The direction of the growth, as well as the configuration of individual cells, is deter- mined in large part by the direction of the fibrin threads which act as support. , In a successful preparation, one may observe the following sequence of events. In the teasing of the fragment, many small thymic cells are often separated from the main fragment, and are then distributed far into the surrounding plasma. The cen- tral bit of tissue soon becomes surrounded by a fringe or halo of isolated cells. Most of these are cells of the lymphoid type, but one may identify coarsely granular cells (eosinophiles?) and scattered myoid cells, the appearance of which in the living has already been described. After a few hours, the isolated small cells sink to the bottom of the plasma. Some however, remain adherent to the cover-glass and migrate out to a considerable distance from the main fragment. As regards the character of the amoeboid activity of the small cells, it is, as Hammar (10) has pointed out, identical with that of the small lymphocytes of the blood, as described by Jolly (11), Askanazy (12), Meves (13) and others. lLobe-like hyaline psuedopodia are extruded and retracted, first from one portion of the protoplasmic margin, and then from another. Often these pseudopodia are long and hair-like, and a number of such delicate filamentous processes, which may reach a length several times the diameter of the thy- mus cell, project from different points of the circumference. When there are active movements of progression, the pseudo- podia of this type seem to be dragged astern like a rudder. The entire cell may become constricted into two lobes and appear to be about to divide, the nucleus changing its contour somewhat with the changing contour of the plasmatic prolongations. The amoeboid activity of certain of the small cells may be maintained for six days or more. Especially at the periphery of the fringe of isolated cells at the bottom of the plasma, one finds many active cells. If a single cell be observed for a pro- 308 ALWIN M. PAPPENHEIMER longed time, one may sometimes note alternating phases of activity and rest; during the latter phase, the cell assumes a spherical shape. Multiplication of the cells does not take place in vitro to an appreciable extent. On but two occasions was actual division observed. One of the cells in a twenty-four-hour culture, was first seen at 8.50 A.M. in a state of active amoeboid motion. Sketches were made at one minute intervals. The cell continued active until 9.47, crawling along the surface of an adjacent large cell. At this time it went into a resting spherical state, remaining inactive until 10.34 a.m. It then again became active, throwing out long pseudopodia and changing its relative position. At 10.45 it again became rounded and motionless. At 10.50 a shallow constriction was first seen dividing the cell into two equal lobes. This rapidly deepened, and at 10.51, the sep- aration was complete, division having taken place in less than two minutes from the first appearance of the constriction. ‘The two cells remained close together until 11.20 when the observa- tion was interrupted. At 1.30 p.m. they had moved apart and could no longer be identified. The second cell came under observation first at 2.29 p.m. Until 3.12 it continued to show active amoeboid motion; at this time it became quiescent, remaining so until 4.01, when a slight constriction was first noted. Division was complete at 4.05 and ten minutes later, one of the new cells became active and moved away from the other. During the division, a slight indication of a spindle was noted, but the chromosomes could not be definitely made out, and the mitotic character of the division could not be established with certainty. In none of the fixed and stained preparations could karyokinetic figures be found, although pic- tures suggesting amitosis were plentiful. The proliferative capac- ity of the lymphoid cells in vitro is therefore a limited one, and multiplication occurs to a negligible extent. The cells retain their vitality, however, for a considerable period, and amoeboid activity has been noted after six days and probably persists even longer. The majority of these cells are of small size and remain indefinitely in an inactive spherical shape. ‘They seem to become denser and more refractile. HISTOLOGY OF THE THYMUS 309 The degenerative changes which occur in the small thymus cells are of two types. There may take place a simple lysis, in which the cells become paler, losing their refractivity and finally appearing as mere shadows. In fixed preparations, the nucleus no longer stains and only a faint outline remains. More com- monly the nucleus becomes smaller and more refractile and stains intensely and diffusely with the nuclear dyes. Occasion- ally, smaller particles are constricted off from the nucleus and extruded from the cells, being found free in the plasma. Whether the reduction in the size of the nucleus is accomplished wholly in this way, is not certain. In fixed preparations of older cul- tures, the majority of the small cells take on this form. The bizarre radial extrusions, which are seen in the mammalian thy- mus under circumstances leading to acute involutional changes, were not often found in the cultures. This is rather surprising, since the small cells of the thymus of infected or starving frogs show these changes in marked degree. Under certain circumstances which were not accurately deter- mined, there takes place an accumulation of fat in the small cells. The most frequent appearance is the presence of three or four rather large fat droplets in the cap of protoplasm corre- sponding to the dell in the nucleus; but scattered droplets may be found anywhere in the rim of protoplasm surrounding the nucleus. In some preparations, almost every cell contained a single larger droplet. No microchemical study as to the nature of this lipoid mate- rial was made. The droplets are highly refractile and stain briliantly with Scharlach R. According to the studiés of Holm- strom (14) and Hart (15) on the rabbit and human thymus, fat droplets, or lipoid granules demonstrable by Ciaccio’s method are normally absent from the small thymic cells; and Ciaccio (16) has found that the lymphoid cells of the blood contain no recog- nizable fat or lipoid substance. Stheeman (17) makes a similar statement in regard to the lymphoid cells of the lymph-nodes. From the above observation, however, one is forced to conclude that the small cells of the amphibian thymus may, under certain conditions, accumulate fat in visible form. Since the fat drops 310 ALWIN M. PAPPENHEIMER - may be present in cells which show active amoeboid motion, and appear otherwise healthy, there is no reason for considering the change a degenerative one, but rather, in accordance with modern ideas, as an evidence of sub-oxidation. In the description of the fixed material, it was stated that granulae could be demonstrated with reasonable certainty in the small thymus cells. The following ‘vital’ stains, applied by adding dilute solutions in Ringer’s fluid to the cultures, showed the presence of granulae; neutral red, trypan-blau, and Janus green. With trypan-roth, isamin-blau, and new methylene blue GG, the refractivity of the protoplasm is so changed that the minutest fat droplets appear with great distinctness, but there is no actual staining of the granules. By adding neutral red to the culture, some but not all of the cells will be found to contain a few granules. It was not however, possible to demonstrate granulae in the small cells by injecting strong solutions into the dorsal lymph-sac, and after general diffusion of the dye had taken place, teasing the thymus in Ringer’s solution. Red stained granules and larger masses of colored material are found in other cells, particularly the myoid cells by this method, as well as when added directly to the plasma. With trypan blau, fine discrete granulae are found in many of the small cells. These are sharply localized to one segment of the cell, forming a dark bluish dise or cap which is very striking when seen with the low power. After a time, the nucleus gradu- ally takes a faint bluish tinge. With Janus green, extremely distinct Ee atiales appear in some but not alleof the cells of the small type. The dark greenish color of the granulae develops slowly, reaching its maximum intensity after about ten minutes. They are seen as sharply circumscribed dots, which vary from barely visible points up to the size of a large coccus. They are grouped at one pole of the cell facing the indentation of the nucleus and show no recogniza- ble radial or linear alignment. They appear to be more numerous and attain a larger size than the granulae seen in Benda prep- arations, but they correspond closely to these as regards their location in the thicker portion of protoplasm facing the depres- sion of the nucleus. Ae i HISTOLOGY OF THE THYMUS dll If a very weak solution of the dye be used, the nucleus remains unstained, but with a stronger concentration takes a distinct purplish-red tinge. It is generally held that nuclear staining takes place in dead cells (Plato (18), Fischel (19), Cesaris-Demel (20)). Since we have no absolute morphological criteria for dis- tinguishing a living from a dead cell, it is probably more accurate to say that staining of the nucleus takes place only in a cell which is dead or injured. The converse is not true, however, and all dead or injured cells do not show nuclear staining with the vital stains.! Now it is certainly possible by means of Janus green to bring about a simultaneous contrast staining of granulae and nucleus in living cells. While I have not observed amoeboid activity in the small cells, nor any other functional indication of life, in the case of other elements which will be described in detail, marked active changes of form took place after staining was completed. The appearance of a reddish color denotes a reduction of the dye- stuff, or in other words a taking up of oxygen on the part of the nucleus, and is in all probability in itself an indication of via- bility on the part of the nucleus. The recent experiments of Warburg and Meyerhof (24) which show that crushed fragments of sea-urchin eggs and even acetone extract powders are capable of taking up oxygen for several hours, perhaps weakens the force of this argument. More convincing is the fact that dead nuclear material which is taken up by phagocytic cells, stains greenish blue and not reddish. 1 To this general rule that staining of the nucleus indicates death or injury to the cell, there are a few exceptions noted in the literature. Thus Przemicki (21), working with certain Protozoa (Opalina, Nyctotherus) succeeded in staining the nucleus in individuals the motility of which was preserved for five days and in which cell division occurred. Goldman (22) in a recent paper states that he has succeeded in vitally staining the nuclei of liver cells. Kite and Chambers (23) announce in a preliminary note that they have produced a differential staining with Janus green of the chromosomes in the spermatic cells of the squash-bug, crickets and grasshoppers, and they have followed the transformation of ana- phase to telophase in a stained spermatocyte. In some observations made by the writer in collaboration with Dr. R. A. Lambert, upon dividing cells of chick embryos in vitro, it was found that the chromosomes were stained red by Janus green, but that division of the cells was arrested upon the addition of the dye. Sk2 ALWIN M. PAPPENHEIMER So far, only the changes occurring in cells of the small type have been considered. But there takes place also a definite out- growth of cells which differ widely from the lymphoid elements and cannot be confused with them. The first evidence of growth is the projection of delicate protoplasinic sprouts from the mar- gin of the main fragment. These have been seen as early as six hours after the preparation of the culture, but they may not appear until the following day, and rarely the first sign of growth is not seen until the second day. Following the appearance of the sprouts, large cells wander out into the medium, either as isolated cells or as coherent tissue-like planes of compact cells. Often these cells take on an elongated spindle shape and arrange themselves in long rows joined end to end, following a fibrin thread or the line of retraction of the plasma. When the cells grow out along the cover-glass, they become flattened, irregularly pyramidal or oval in shape, with long dendritic, barely visible plasmatic processes uniting them to the central fragment or to each other. The individual cells, as seen by the figure (fig. 7) are often of extremely large size, but are so irregular in shape that it is difficult to give measurements of value. The nucleus is relatively large, elliptical, though sometimes indented by large fat droplets in the cytoplasm. It is usually possible to distin- guish one or two slightly more refractile nucleoli; otherwise, the nuclear substance appears homogeneous. From their first appearance these cells are found to be filled with numerous granulae. At first, these are of small and uni- form size, and but moderately refractile; after several days of incubation, the cells contain in addition, many droplets of vary- ing size, which are refractile and evidently fat droplets. The smaller droplets or granulae often range themselves in rows of considerable length. The cell processes are relatively free from granulae, but occasionally do contain small granules, or larger fat droplets. One frequently sees knob-like thickenings along the coarse of a long plasmatic process in which such granules are found. Where the prolongation of the cell ends blindly, the termination is frayed into delicate hair-like processes, which because of their slight refractivity, appear to shade off into the plasma. ~_—-- 2, HISTOLOGY OF THE THYMUS 35113} Often a fibrillar structure of the cytoplasm is recognizable in the living cell, the fibrils being especially distinct in the long slender protoplasmic processes. ‘The appearance of these cells and their usual manner of growth, is indicated in figures 5, 6 anid: 7: Changes of contour and relative position are readily detected in these cells, if the observation be sufficiently prolonged. They do not, however, under normal conditions, show as rapid changes of shape, as do the cells of the lymphoid type. After the first days of active emigration and growth, they retain their position and outline unchanged for many days, gradually accumulating fat drops in their protoplasm. An interesting phenomenon which was seen in the living cells, and confirmed by subsequent fixation and staining, is the sepa- ration of portions of protoplasm, which gradually become con- stricted off and are set free into the plasma. The separated portions contain fat drops and granulae. The process resembles curiously the formation of blood platelets from megakaryocytes, - as first described by H. Wright (25). Its significance here is uncertain; it may be found in cells which show no other degen- erative changes. The stained preparations of these cultures give somewhat vary- ing pictures according to the method of fixation. After formalin, followed by iron-hematoxylin (Heidenhain), the nuclear sub- stance stains homogeneously, the intensity depending upon the extent of decolorization. There are one or two nucleoli of large size, deeply stained, but often with a slightly paler center. There are in some of the nuclei, minute granules surrounded by a clear halo. The cytoplasm shows a beautifully reticulated structure, being composed apparently of delicate fibrillae, occasionally parallel in their course, but disarranged by the presence of smaller and larger fat vacuoles. Along the meshes of the reticulum are deeply staining granulae of smaller and larger size. Most of them are larger than the granules of the eosinophile cells. They are usually spherical, but may be slightly elongated, or when in apposition to a large fat globule, crescentic. The numbers vary; 314 ALWIN M. PAPPENHEIMER in some cells closely aggregated, in others less numerous. They may extend out into the cell processes; usually there is a narrow zone at the surface of the cell which contains fewer granulae. They are often grouped in linear alignment about a fat vacuole, or in the long axis of the cell and especially in the protoplasmic processes where the protoplasmic fibrils run parallel. The gran- ulae do not always stain with the same intensity in the same cell, but this may be due to uneven penetration of the stain or decolorizing agent through the plasma (figs. 5 and 6). In most of the cells there is an area adjacent to the nucleus where the protoplasm is denser and the granulae and fat. drops are absent. Although centrosome or cytasters were not seen in these preparations, it is probable that this denser granule-free area represents the cytocentrum. When the cultures are fixed in bichloride-acetic acid, the eyto- plasmic granulae are very indistinctly brought out by the Heiden- hain stain. The nucleus on the other hand, instead of appearing ‘ homogeneous as in the formalin preparations, shows finely dis- tributed chromatin clumps. The only mitotic figures seen in these cells during the course of the work, were in a specimen fixed in bichloride-acetic acid. The chromatin threads of the dividing cells (prophase and diaster stage) were very large and distinct. The cytoplasmic granules of the cells were also demonstrated by the Altmann-Bensley method, the loosening and retraction of the clot being prevented by preliminary fixation in 10 per cent formalin. The stained preparations however were unsatisfactory since the decolorization of the fuchsin in the plasma clot was impossible, and only a few cells which by fortunate chance, lay within the retraction zone, were available for study. By this method, smaller rounded cells filled with fuchsinophile granules were also seen. The granulae in part show swelling and disintegration, but may persist in this altered form even after the cell has degenerated. The granulae of the large cells may be brought out deel by the use of vital stains, the most successful of those tried being Janus green and methylene-blue GG. With the former stain, HISTOLOGY OF THE THYMUS alo the granulae gradually assume a dark greenish color, reaching its maximum intensity after eight or ten minutes. The concen- tration of the dye appears to have slight influence upon the rate of staining, but as in the case of the small cells, concentrated solutions produce a reddish staining of the nucleus. The smallest granulae appear to stain first and most intensely, and transitions of every degree are noted up to the large refractile unstained fat drops. The largest droplets often have a pronounced greenish tinge in certain focal planes; whether this is due to refraction of the color from surrounding stained granules, or whether the fat globules are enclosed in a stained shell, could not be decided. Without entering into a discussion of the réle of the cell granulae in the synthesis of fat, it may be said that the appearance noted rather suggests the direct transformation of the granules or a portion of them into fat. Not only are there apparent tran- sitions between the stained granules and the larger refractile droplets, but in Sudan preparations, the fat droplets may be of extremely small size, and distributed in the same linear align- ment as the normal granulae. An attempt was made to examine more closely into the relation of the granules to the fat drops, by fixing the vitally stained cell, and subsequently staining with Sudan III; but it was found that the Janus green was rapidly decolorized by the formalin. As the staining with Janus green progresses, there occurs a remarkable contraction of the entire cell. The long plasmatic prolongations are shortened, thickened and gradually withdrawn into the cell body. The entire cell becomes plumper and tends to assume a globular shape, the granulae and fat drops becoming clumped about the nucleus. The phenomenon may occupy only a few minutes. After having taken on a spherical shape, the cell for a time extrudes rounded ectoplasmic pseudopodia in various directions. This takes place even when the nucleus is distinctly stained reddish by the dye, and as has been said, this affords an example of nuclear staining in a cell which though injured, still shows vital activity. Gradually these amoeboid extrusions cease, and the cells remain indefinitely in a globular form. The staining, however, fades THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO. 3 316 ALWIN M. PAPPENHEIMER completely within twenty-four hours, the cell retaining only a faint diffuse greenish tinge. If the Janus green be added originally to the plasma in which the tissue is implanted, no growth or emigration of cells occurs. This is probably due to a toxic effect of the stain as well as to an inhibition of amoeboid activity.” With new methylene blue GG, in very dilute solutions, there is also produced a very sharp blue staining of the cytoplasmic eranules. The nucleus remains unstained, and retraction of the cytoplasmic processes does not occur to any extent. The small thymic cells take a pale diffuse bluish tinge, but the granules though made visible by the altered refractivity, do not them- selves stain. Before discussing the origin of these large growing cells, men- tion should be made of their power to phagocyte the small thymic cells. Both in living and fixed preparations, the presence within the large cells of more or less intact small cells is easily recog- nized. Often the nucleus of the ingested small cell stains intensely with the vital stains, such as the new methylene blue, when the majority of the extra-cellular small cells are unstained. It may be fairly assumed from this that the ingested cell is dead. Such staining of the phagocyted cell does not always occur, and the same phagocyte may contain both stained and unstained nuclei of the small cell type. Morphologically, the ingested cell may show no degenerative change save a condensation and increased refractivity of the nuclear material. Stainable granulae may be found in considerable numbers in these phagocytic cells, distributed between the ingested small cells, and fat droplets. This observation is not in accord with the statement of Schulemann (26), that cells which have used up their ‘receptors’ in the process of phagocytosis no longer give 2 Through the kindness of Professor Wherry, who supplied me with a culture, it was possible to try the effect of the dilute solutions of Janus green upon the motility of Amoeba limax. Amoeboid activity was completely inhibited after a few minutes, the amoebae taking on a globular form. A control on the same slide, to which a drop of Ringer’s solution was added, remained actively motile after twenty-four hours. « HISTOLOGY OF THE THYMUS ole a vital staining of the granules with trypan-blau. He found that macrophages from lymph nodes, when filled with erythro- cytes, no longer contained stainable granulae. In the cells under consideration, the phagocytosis of other cellular elements is not accompanied by a disappearance of the granules. There occurs then, in plasma cultures of frog thymus, a growth and to a limited extent, a multiplication of large cells of varying morphology, but evidently identical origin. The growth may be in the form of fairly compact, tissue-like sheets of cells, in a loose anastomosing reticulum, in long chains of cells joined end to end, or the cells may be entirely isolated, rounded or with plasmatic prolongations of varying tenuity and length. All types of growth and transitions between them may be seen in one and the same culture. The character of the nucleus in all the cells is essentially the same, but its contour is naturally mod- ified with the varying contour of the cell body. The cells all contain granulae ranged upon a fibrillar ground-work, and demon- strable both in vitally stained and in fixed preparations by a variety of methods. The cells show from the first a tendency to accumulate fat, and after this has reached a certain grade, usually by the fourth or fifth day, further growth is retarded or checked. The cells may be phagocytic towards the small thy- mus cells. The probable nature and origin of these cells were not easy to establish. Three possibilities suggested themselves: (1) that they were derived from connective tissue cells, either from the capsule or from the septa accompanying the blood vessels; (2) that they were derived from the endothelial cells of the capil- laries; or (8) that they were outgrowths of the epithelial reticulum of the gland. The last view was the one finally adopted and for the following reasons. ‘The growth could never be traced to the capsule of the gland when portions were incorporated in the tissue fragment. The connective tissue of the capsule showed but shght capacity for growth, only a few fibrillated spindle cells occasionally penetrating the clot for a short distance. Some- times portions of striated muscle and connective tissue were included in the fragment, but there was never any outgrowth of 318 ALWIN M. PAPPENHEIMER cells of this type, nor from pieces of spleen, heart muscle or intestinal wall used as controls. That these cells were derived from capillary endothelial cells seemed improbable. The manner of their growth and their fre- quent origin from small masses of cells in which the absence of capillaries could be determined with certainty, seems to exclude this possibility. More positive evidence in favor of their origin from the reticular cells, is that they may, in thin portions of the culture, resemble closely the normal protoplasmic cellular framework of the gland. Although the protoplasm shows a fibrillated structure, evident especially in fixed material, there is no formation of definite fibrils upon the surface of the cells, nor do the plasmatic processes of the cells, no matter how long- drawn-out, resemble the more rigid and refractile processes of the growing connective tissue cells. In their power of phago- cyting the small cells, they function as do the normal reticular cells of the thymus. But since other cells in vitro may assume phagocytic powers, too much emphasis should not be laid upon this point. If the assumption be correct that the growing cells of these cultures are derived almost wholly from the reticular cells, then we must, if we accept the prevailing view as to the histogenesis of these elements, hold them to be epithelial in nature. The epithelial origin of the thymic reticulum is believed in by all the recent workers on the structure of the thymus, including Hammar (27), Stohr (28), Schridde (29), Maximow (30), and Cremieu (31). Dustin (32) and Pigache and Worms (33), amongst recent writers, still hold to the old view that the thy- mus, like the lymph-glands, has a fibrous reticulum.? Salkind (34), in a recent paper, takes an intermediate position, claiming to have demonstrated by special methods, a fibrous reticulum analogous to that of lymph-glands, coexisting with the epithelial reticulum and giving origin to the lymphoid cells. If his views, 3 For a complete discussion of the histogenetie origin of the thymus elements, the reader is referred to the exhaustive reviews of Hammar (27) (Ergebnisse d. Anat. u. Entwick., 1910, Bd. 19, p. 1) and of Wiesel (35) (Lubarsch and Ostertag’s Ergebnisse d. Allg. Path. u. Path. Anat., 1911, Bd. 15, 2, p. 416). ee Oe a :?. HISTOLOGY OF THE THYMUS 319 which are counter to the prevalent conceptions of the thymic reticulum, prove correct, the interpretation of these growing elements as epithelial in nature, would be rendered less probable. The cells in question might then be derived either from the fibrous or the epithelial reticulum. Nevertheless, it is certain that in the mammalian thymus at least, it is impossible to put in evidence such a fibrous reticulum, either with Mallory’s ani- line blue method or with the silver impregnation method of Bielschowsky, both of which bring out with great clearness the reticular fibers of the lymph nodes. Until Salkind’s work re- ceives further confirmation, it seems unwise to revise again the current and well founded conception of the exclusively epithelial origin of the thymus reticulum. Much emphasis has been laid by Lambert and Hanes (36) upon the differences in the character of in vitro growth of epi- thelium and connective tissue. The former tend to form solid coherent growths, while in the latter, the cells, though often connected by plasmatic processes, tend to remain isolated from one another. As a general distinction, this has undoubtedly proven to be true. The tissue which we have been studying forms at times an apparent exception since, though epithelial, it may grow either in coherent sheets of closely apposed cells, or as a loose anastomosing network, or indeed, these cells may lose their plasmatic connections with adjacent cells and wander isolated far out over the surface of the plasma. This variability in the manner of growth is not surprising if we recall the normal development of the parent tissue, and the changes which it undergoes. Beginning as a solid outgrowth of cells, the thymic epithelium early becomes rarefied into a protoplasmic syncytium in the meshes of which lie the small cells. It is probable also that under certain abnormal conditions, the reticular cells of the mammalian thymus assume a rounded form and become rela- tively independent of their cellular connections, often, too, exer- cising a phagocytic function upon the small thymic cells (Rud- berg (37), Pappenheimer (7), Cremieu (31) and others). That the thymic epithelium should differ widely in the manner of its growth from the epithelium of glandular organs or of solid epi- 320 ALWIN M. PAPPENHEIMER thelial tumors, might be expected. It is nevertheless interesting that the tissue growing under highly artificial conditions, should conform so closely to its normal type, and in the phagocytosis of the small cells, exercise a function which is characteristic of the normal thymic reticulum. Before discussing the conclusions to be drawn from the fore- going observations, I wish to record briefly certain further studies on the comparative growth of thymus and lymph-nodes. The tissues for these experiments were obtained from young adult rats and incubated at 37°. The small thymic cells emigrate rapidly, often reaching the edge of the plasma drop within a few hours. The lymphocytes of the lymph glands behave in the same way. Degenerative changes begin rapidly, both in the thymic cells and in the lympho- eytes. The nuclei become pyenotic, fragment, and finally break up into globular, deeply staining particles. Well preserved, actively amoeboid cells were not found after forty-eight hours. Growth from the thymic fragments begins usually on the second day, as fusiform or polygonal cells with intercellular con- nections. Large flat cells on the cover-glass resemble those described in the frog’s thymus; but there is frequently a radially directed growth of long spindle cells resembling connective tissue. After three or four.days, however, there is usually a tendency towards the formation of flat cellular planes, the growing margin of which is very definite and sharply limited. A few cells at the periphery may become partially or completely separated, but the growth on the whole is coherent (fig. 8), and often ap- | proaches in its character the growth of epithelial tissue from carcinomata, as described by Lambert and Hanes (36) and L. Loeb (88). At this stage of the growth, moreover, the thymus culture shows changes which distinguish it definitely from the culture of lymph-node. The central fragment becomes rarefied, and there appear large numbers of globular cells of large size, which with the low power seem filled with coarse granules. These granules are really ingested small cells in various stages of pyc- nosis and degeneration. Scattered phagocytic cells of this type HISTOLOGY OF THE THYMUS 321 appear among the growing cells at the margin. Where these lie against the cover-glass, they may retain their irregular shape and plasmatic processes, but the majority are globular (fig. 10). Transitions between these phagocytic cells and the growing healthy cells are evident, and their origin from the reticular cells could be definitely proven by studying serial sections of the growing tissue. The lymph-nodes also show a growth of the fixed elements begining usually on the second or third day, and almost invari- ably as radially directed sprouts. As the growth proceeds, the fragment becomes surrounded by a halo of spindle or stellate cells, which resemble in all respects, growing connective tissue cells from other organs (fig. 9). As compared with the growing reticular cells of the thymus, the cell processes are often more numerous and rather of the dendritic type with secondary branch- ing. There is often a fibrillar differentiation on the surface of the protoplasm. The nuclei are smaller than those of the thy- mic cells and stain more intensely. Rarefaction of the ceatral fragment does not occur, or at least not until a much later stage of growth. It is never so pronounced as in the thymic cultures. While it is occasionally possible to find a few lymphoid cells enclosed within a larger connective tissue cell, phagocytosis is far less conspicuous than in the thymus, and large globular cells stuffed with ingested small cells are never seen. Either the reticular cells of the thymus possess phagocytic properties which the lymph gland reticular cells do not have to the same degree; or the small thymic cells are more susceptible to phagocytosis than the lymphocytes of the lymph gland. The former expla- nation is the more plausible. The behavior of the small cells in thymus and lymphoid tissue, has been found to be the same in all other respects; whereas the difference in structure and origin of the reticular cells in the two tissues makes a difference in function the more probable. 322 ALWIN M. PAPPENHEIMER CONCLUSIONS Minute granulae of a type not hitherto described, were demon- strated in the frog’s thymus, by the use of Benda’s mitochondrial method. Larger gentianophile granules and droplets were found in some of the cells of this type. Whether these were secretory or degenerative in nature was not determined. Granulae, possibly of the same nature as those demonstrable by the mitochondrial methods, were shown to be present in the living cells by the use of vital stains. The small thymic cells also contain granulae, and in this respect, the small thymus cells are identical with the lymphocytes of the blood. This observation is in direct opposition to that of Schridde (29), that the small thymus cells, which he believes with Stohr (20) to be of epithelial origin, may be differentiated from true lymphocytes by the absence of granulae. In clotted plasma cultures, there is a radical difference in the behavior of the small and large thymus cells. The former show practically no capacity for further proliferation, but after a period of active motility, undergo degeneration; the latter exhibit active growth, often in the form of syncytial cell masses. They are actively phagocytic towards the degenerating small thymus cells. This characteristic difference in the behavior of the two Sve of cells is opposed to Stéhr’s view that the small cells are modi- fied epithelial reticular cells and that transitions between the two normally occur. ; The small cells of the rat thymus show absolutely no morpho- logical differences from the lymphocytes of the lymph nodes; they exhibit the same active motility and the same proneness to undergo degeneration when kept in vitro. The growth of rat thymus differs from that of lymph nodes (1) in the early rarefaction of the implanted fragment, with the appearance of numerous large phagocytic cells; (2) in the for- mation of tissue-like planes composed of epithelial reticular cells differing in their appearance from the fusiform or stellate cells which grow from the connective tissue capsule or reticulum of HISTOLOGY OF THE THYMUS 323 the lymph-nodes. This characteristic difference corresponds to the different histogenesis of the thymic reticulum and suggests a different function. In conclusion, the writer wishes to express his obligation to Dr. E. G. Kite for his kindness in supplying the vital stains used in this work, and to Dr. R. A. Lambert for his assistance in the preparation of the rat tissue cultures. 324 ALWIN M. PAPPENHEIMER LITERATURE CITED (1) Benstey, R. R. 1911 Studies on the pancreas of the guinea-pig. Am. Jour. Anat., vol. 12, no. 3, p. 389. (2) Mreves, F., anp DursserG, J. 1908 Die Spermatocytenteilungen bei d. Hornisse. Arch. f. Mikr. Anat., Bd. 71, p. 571. (3) Mayer, S. 1888 Zur Lehre v. D. Schilddriise u. D. Thymus bei den Amphibien. Anat. Anz., Bd. 3. (4) Pensa, A. 1902 Osservazione a proposito di una particolarita di struttura del timo. Boll. della Soc. med.-chir. di Pavia. 1904 Ancora a proposito di una particolarita di struttura del timo negli anfibi Anuri, ibid. 1905 Osservazione sulla struttura del timo. Anat. Anz., Bd. 26. (5) WEISSENBERG, R. 1907 Ueber die Quergestreiften Zellen d. Thymus Arch. f. Mikr, Anat., Bd. 70. ' (6) Hammar, A. 1905 Histologie u. Involution d. Thymus. Anat. Anz.,Bd. Be : (7) PApPpENHEIMER, A. M. 1910 A contribution to the normal and pathologi- cal histology of the thymus gland. Jour. Med. Research, vol. 17, p. 1. (8) Prenant, A. Contribution 4 1’étude organique et histologique du thymus, etc. La Cellule, T. 10. (9) Foor, N. 1912 Ueber das Wachstum von Knochenmark in Vitro, ete. Beitr. z. path. Anat., Bd. 53, p. 446. (10) Hammar, A. 1907 Ueber die Natur der kleinen Thymus-zellen. Arch. f. Anat. u. Entwick.. Bd. 83. (11) Jouny, J. 1903 Surles mouvements des lymphocytes. Arch. de méd. exp., T. 15, p. 54. (12) Askanazy, M. 1905 Ueber améboide Bewegung d. Lymphocyten. Ctlblt. f. allg. Path., Bd. 16, p. 897. (13) Mrves, F. 1910 Zur Einigung zwischen Faden- u. Granula Lehre des Protoplasma, etc. Arch. f. Mikr. Anat., Bd. 75 p. 642. (14) Hotmstr6m, R. 1911 Ueber das Vorkommen von Fett u. fettahnlichen Substanzen im Thymusparenchym. Arch. f. Mikr. Anat. Bd. 77, p. 323. (15) Harr, K. 1912 Ueber das Vorkommen v. Fett ind. Thymus. Die patho- logische Involution d. Thymus. Virch. Arch., Bd. 207, p. 27. (16) Craccto, C. 1910 Sul lipoidi dei leucocyti. Fol. Clin. Chim. Mier. vol. 2. (Abstr. in Ctrlblt. f. Bioch. u. Biophys., vol. 10, p. 551.) (17) Srureman, H. A. 1910 Histologische Untersuchungen tibdr die Beziehun- gen des Fettes zu d. Lymphdriisen. Beitr. z. path. Anat. Bd. 48, p. 170. (18) Puaro, J. 1905 Ueber die vitale Farbbarkeit d. Phagocyten, ete. Arch. f. Mikr. Anat., Bd. 56, 468. (19) ,FISCHEL, A. 1901 Untersuchungen iiber die vitale Firbung. Anat. Hefte, vols. 52, 53, p. 415. (20) Crsaris-Demrz, A. 1909 Ueber d. Morphologische Struktur u. d. morph. u. chromat. Veranderungen d. Leukocyten. Virch. Arch., Bd. 195, p. 1. (21) Przemicxr, A. M. 1897 Ueber die intravitale Farbung des Kernes u. des Zellprotoplasmas. Biol. Ctrlblt., Bd. 17. HISTOLOGY OF THE THYMUS 325 (22) GoutpMAN, E. 1912 Vitale Farbung u. Chemotherapie. Berl. Klin. Woch., no. 36, p. 1689. (23) Kite, G. L., AnD CHAMBERS, R., Jr. 1912 Vital staining of the chromo- somes and the function and structure of the nucleus. Science, N. S. vol. 36, p. 639. (24) Warsurc, O. AND Meyernor, O. 1912 Ueber Atmung in abgetéteten Zellen u. in Zellfragmenten. Pfliiger’s Arch. f. d. ges. Phys., Bd. 148, p. 293. (25) Wricut, J. H. 1906 Die Entstehung d. Blutplittchen. Virch. Arch. Bd. 186, p. 55. (26) ScouLEMANN, W. 1912 Beitrage z. Vitalfairbung. Arch. f. Mikr. Anat. Bd. 79, p. 223. (27) Hammar, A. 1910 Ergebnisse d. Anat. u. Entwick. Bd. 19, p. 1. (28) St6éHR, P. 1906 Ueber die Natur der kleinen Thymus-elemente. Anat. Hefte, Bd. 31, p. 408. (29) ScurippE, H. 1909 Pathologische Anatomie, Bd. ii, L. Aschoff, Art. Thy- mus, 8. 148. Jena. (830) Maximow, A. 1912 Untersuchungen iiber Blut u. Bindegewebe. IV. Uber die Histogenese d. Thymus bei Amphibien. Arch. f. Mikr. Anat. Bd. 79, p. 560. (31) Crimiev, R. 1912 Etude des effets produits sur le thymus par les rayons X. Lyon, Imprimeries Reunies. (32) Dustin, A. P. 1911 Le thymus de 1l’Axolotl. Arch. de. Biol, T. 26. (33) Piaacure, R. et Worms, G. 1910 Les degénerescences cellulaires du thy- mus. Bull. et memoires Soc. anat. de Paris. T. 85, p. 854. (34) Sauxinp, J. 1912 Sur Vorganization du thymus. Note preliminaire. Anat. Anz.. Bd. 41, p. 145. (35) WreseL, J. 1911 Lubarsch u. Ostertag Ergeb. d. Allg. Path. u. Path. Anat., xv/2, 416. (36) Hanus, F. anp LamMBertT, R.A. 1912 Amédboide Bewegung von Krebszellen als en Faktor des invasiven u. metastatischen Wachstums maligner Tumoren. Virch. Arch. Bd. 209, p. 12. (387) RupBere, H. 1907 Studien iiber die Thymusinvolution. I. Die Involu- tion nach Réntgenbestrahlung. Arch. f. Anat. u. (Phys.) Suppl. (38) FLerisHer, M. S. anp Lozs, L. 1911 The relative importance of stroma and parenchyma in the growth of certain organs in culture media. Proc. Soc. Exp. Biol. Med., vol. 8, p. 1383. PLATE 1 EXPLANATION OF FIGURES ‘L Benda fixation and stain. Mitochondria, and fibrils of myoid cell. X 1000; camera lucida. 2 Benda fixation and stain. Granulae of small thymus cells. > 1000. 3 Epithelial cell complex from medulla. Granulae and droplets. Benda fixation and stain. X 1000. 4 Cell complex from medulla, containing granulae and irregular masses of gentianophile substance. Benda fixation and stain. X 1000. 326 HISTOLOGY OF THE THYMUS PLATE 1 ALWIN M. PAPPENHEIMER 7327 THE AMERICAN JOURNAL OF ANATOMY, VOL, 14, NO. 3 PLATE 2 EXPLANATION OF FIGURES 5 Tissue culture, eight days. Large spindle cell with granulae and ingested small cell. Formalin, Heidenhain. X 1000. 6 Tissue culture, eight days. Large reticular cell, with granulae. Formalin, Heidenhain. > 1000. 7 Forty-eight hours growth in vitro. Large reticular cells on cover-glass. 125. 328 HISTOLOGY OF THE THYMUS ALWIN M. PAPPENHEIMER 329 PLATE 2 PLATE 3 HISTOLOGY OF THE THYMUS ALWIN M. PAPPENHEIMER Ase Oaare @ eo Vag. Px PS : ae iN 0) > - 9 & EXPLANATION OF FIGURE 8 Rat thymus; four days growth in vitro 330 HISTOLOGY OF THE THYMUS PLATE 4 ALWIN M. PAPPENHEIMER EXPLANATION OF FIGURE 9 Rat lymph gland; four days growth in vitro ymph g ) 301 PLATE 5 HISTOLOGY OF THE THYMUS ALWIN M. PAPPENHEIMER \ EXPLANATION OF FIGURE 10 Rat thymus. Large reticular cell, filled with ingested small thymus cells and nuclear fragments. 332 THE DEVELOPMENT OF THE ELASMOBRANCH LIVER I. THE EARLY DEVELOPMENT OF THE LIVER Il. THE DEVELOPMENT OF THE LIVER DUCTS AND GALLI-BLADDER RICHARD E. SCAMMON From the Institute of Anatomy, University of Minnesota FIFTY-FOUR FIGURES CONTENTS PART I. THE EARLY DEVELOPMENT OF THE LIVER WeelonGrodwctlonmens. 05 eee: J PRA MER IEICE De Bet. Soce eum eae SOOT 333 GIPRUGE Tray G Ure tae | RIE oh ccc, i seena cs steclligs Crea aw TR th a aie. aie tases ay aera 334 III. Development of the liver in embryos from 3.0 to 10.0 mm. in length. ... 338 Ne @ om CSO See bec) Sic Fite gs SE Ie Ce I ey eaers« Oh.0 4 a Se ae bale 350 PART IJ. Tur DEVELOPMENT OF THE LIVER DUCTS AND GALL BLADDER I. Description of fully formed biliary apparatus........................ 356 II. Development of the hepatic ducts and their rami.................... 359 III. Development of the gall bladder and cystic duct..................... 376 LV. Development of the ductus choledochus... . ¢ 2... 5 0:47 022% 4. domes nee sae 380 Veg IG Mm (TEL aN ee to es Se 386 Eaton pen ayp lyase tant ett sah Sat. Lids s o< aay Sys ON eae em rane erage oc voi A 390 I. INTRODUCTION The development of the elasmobranch liver offers many problems of interest not only for themselves, but because of their bearing upon questions regarding the structure of this organ in the vertebrates in general. The hepatic duct system shows here in its earlier stages a number of characters which are masked in forms which are more complicated or which undergo a more rapid development and in later stages the organ exhibits a num- ber of peculiarities of interest in the light of recent work on the relation of parenchymous to vascular structures. 333 THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO. 3 334 RICHARD E. SCAMMON The present paper, based upon a study of an extensive series of embryos of Squalus acanthias' gives an account of the early development of the liver and the history of the principal liver ducts and the gall bladder. In a later section it is hoped to give an account of the development of the smaller rami of these ducts and of the hepatic parenchyma. Il. LITERATURE At this point I shall review only briefly the literature of the general development of the elasmobranch liver. Particular points are considered in more detail in the separate sections and reviews of the literature by Brachet (97), Choronschitzky (’00), Piper (02), and Weber (’03) already cover a part of the subject. In common with so many points in selachian embryology there was but little knowledge of the development of the liver until the researches of Francis Balfour. Rathke (’27) published an account of several selachian embryos including one of Squalus mustelus (Mustelus canis?) of an approximate length of 45mm., in which he described the division of the liver into an anterior mass and two posterior lobes and traced the course of. the ductus choledochus to the intestine. He stated that the gall bladder was absent in this specimen as well as in an older one of Squalus canicula (Seyllum eanicula’?). At such a stage this structure is, in fact, embedded in the liver substance and not visible exter- nally. Rathke observed the gall bladder however in an embryo of Squalus mustelus 7 inches 2 lines in length, and traced the course of the vitelline veins to the liver and followed their ramifications in this organ to their final connections with the sinus venosus. Franz Leydig (’52) in his “Beitrage zur microskopischen Anatomie und Entwicklungsgeschichte der Rochen und Hai” ‘This material consisted of sectioned embryos of Squalus acanthias from 3 to 86 mm. in length as well as several specimens of the same species in the ‘pup’ stage, and a few large embryos of Mustelus laevis and Squalus sucklii (?).. Ina large part these specimens were from the Harvard Embryological Collection, and I wish to express here my thanks to Dr. Charles 8. Minot for their use for a prolonged period, as well as for the privileges of his laboratory during a part of the time while this study was in progress. Four specimens were also from the embryological collection of the University of Kansas. For their use I am indebted to Dr. C. E. McClung. DEVELOPMENT OF THE ELASMOBRANCH LIVER 335 figured a cleared embryo of Squalus acanthias approximately 18 mm. in length in which the liver is pictured as a dark irregular mass lying just posterior to the heart. He described the cells of this organ as arranged in lobules and containing numerous fat droplets embedded in a homogeneous ground substance. He | also traced the course of the omphalo-mesenteric veins through the liver. Balfour (76) working with Scyllium, Pristiurus and Torpedo, described the liver as arising in Stage I, when forty-eight’pairs of somites and three pairs of gill pouches are present, as a ventral outgrowth from the ‘duodenum’ directly anterior to the umbilical canal. This outpouching gives off at once two lateral diverticula which are the rudimentary lobes of the liver, while the remainder of the original ventral median pouch forms the gall bladder and ductus choledochus. The hepatic tubule diverticula appear as hollow buds by stage K, and increasing rapidly both in length and number soon anastomose forming a regular network. In the course of these changes the lumina of the tubules become much reduced in size. The gall bladder arises as dilatation of the anterior end of the median pouch and its duct Joining with the hepatic ducts forms the ductus choledochus. Hammar (’93) figured and described the first series of recon- structions of the selachian liver. These are of embryos of Tor- pedo ocellata, the first of forty segments, and the remainder, 9, 11, 15 and 18 mm. long respectively. These specimens corre- spond roughly to Balfour’s stages J, K, L, M, and O. From the study of these models Hammar concludes that the liver arises primarily from three diverticula, two lateral and one median in position, and not from a single median and ventral pouch as stated by Balfour. He also considered the liver proper to arise from the lateral diverticula, the median giving rise to the gall bladder and its duct only. He noted further that there was a twisting of the fore gut from left to right, a point apparently overlooked by other observers. The account of the formation of the common bile duct and gall bladder is fairly complete, but he traced the hepatic ducts no farther than their entrance into the lateral masses of hepatic trabeculae. 336 RICHARD E. SCAMMON In a later paper of a more general nature on the early develop- ments of the liver, Hammar (’97) expresses his views in regard to the origin of the selachian liver as follows: Bei den Selachiern wird ebenfalls eine unter dem Herzen hervorra- - gende stufenihnliche Leberfalte gebildet, an deren cranialen Rand schon frihzeitig zwei bilateral-symmetrische Divertikel auftreten—Zwischen diesen beiden Divertikeln und beinahe gleichzeitig mit ihnen entsteht als eine cranioventrale Verlingerung der Leberfalte noch ein drittes medianes Divertikel, aus welchen die Gallenblase und Gallenblasengang hervorgehn.? I quote Hammar at length for he holds a view somewhat differ- ent from that accepted by most investigators and one which this paper will in part confirm. Laguesse (’94) gave a brief account of the development of the liver in Squalus acanthias in connection with his study of the pancreas in this form. He states that the liver arises a little later than the pancreas, a point which has since been disproven, and although in possession of younger embryos, he apparently first observed the organ in an embryo 8 mm. in length, where it appeared as a thick walled ventral pouch extending from the primitive sinus venosus to the anterior wall of the yolk-stalk. An embryo of 9 mm. length showed the formation of the lateral diverticula. At 16 mm. the buds of the hepatic tubules had appeared and at 19 mm. they were fused together, forming the typical net-work of hepatic trabeculae so often described. La- guesse emphasizes the late appearance of the gall bladder as a structure distinctly separated from the hepatic anlage proper. This seems to me to be a point of much importance which apparently has not been recognized by other workers in this field, with the exception perhaps of Hammar. Brachet (’96) devoted the first part of his contribution to the development of the liver and pancreas to the selachian liver as represented by Torpedo ocellata. Like Hammar he presented a series of reconstructions corresponding to Balfour’s stages J, K and L. The two main points in this paper consist of, first, an affirmation of Balfour’s statement that the liver arises between 2 The italies are the author’s. pers DEVELOPMENT OF THE ELASMOBRANCH LIVER aan . the anterior intestinal portal and the sinus venosus as a single median ventral pouch from which the lateral pouches secondarily arise, and second, the recognition of the fact that the medita pouch which can be distinguished when the liver reaches the rin- lobed stage consists of two portions, named in accordance with the nomenclature suggested by Goeppert (’93) the ‘pars hepatica’ which lies anteriorly and Joins with the two lateral pouches in forming the true hepatic parenchyma, and a posterior portion called the ‘pars cystica’ which forms the gall bladder and the cystic and common bile ducts. Brachet followed the history of the latter structures in detail, but gives little information as — to the history of the hepatic ducts, although he recognized that they were formed from the bodies of the lateral pouches and con- sidered that these pouches were reduced in caliber in the course of their transformations. In the Ergebnisse for the same year Brachet (’97) repeats his conclusions and summarizes the preced- ing literature since the time of Balfour. Riickert’s work (’96) on the development of the spiral valve in Pristiurus is illustrated by three reconstructions, two of which include the gall bladder and ductus choledochus and illustrate well the forward migration of the former structure. Riickert describes the migration of the ostium of the ductus choledochus in relation to the vitelline duct and its movement from left to right along with the spiral valve in later stages. ; Mayr (97) in his account of the development of the pancreas in Pristiturus and Torpedo incidentally describes the condition of the liver in several embryos. His description coincides with that of Balfour and Brachet, but he emphasizes the fact that in early stages the pars hepatica is single anteriorly and that the two lateral pouches diverge from the median line as they extend backward. Choronschitzky (00) published a paper of some magnitude, describing the development of the liver and certain other viscera in all classes of vertebrates. Torpedo was employed as a repre- sentative of the fishes. He gave an account of four stages of this form, the youngest being one in which the liver consisted of a median and two lateral pouches from which four ‘secondary’ 338 RICHARD E. SCAMMON pouches sprang and the oldest one in which the liver had reached . a length of 1.1 mm. as measured from transverse sections and was a solid parenchymous organ. He traced the separation of the liver from the gut and the formation of the gall bladder in some detail and followed out in a general way the development of the two larger hepatic ducts. On the whole his description is a confirmation of that of Brachet. The reconstruction method was apparently not employed. The work of Debeyre (’09) is primarily a study of the origin of the hepatic cylinders. However, working on Laguesse’s Acan- thias material, he confirms that author’s account of the early stages of the liver, including the location of the gall-bladder anlage in the extreme posterior end of the hepatic diverticulum. The papers of Braus (’96), Holm (’97), and Minot (’00) deal mainly with the histogenesis and vascularization of the liver, but contain incidental references to its early development. Braus and Minot accept Balfour’s account as essentially correct. III. DEVELOPMENT OF THE LIVER IN EMBRYOS FROM 3 TO 10 MM. IN LENGTH In Acanthias the first evidences of liver development are to be seen in embryos of 19 to 21 segments. At this stage, which is a little younger than No. 16 of the Normal plate series and lies between Balfour’s stages G and H, the embryo shows a distinct head bend and the medullary canal is closed except for the large netiropore. The archenteron which is still’ in quite a primitive condition is outlined in figure 1, a graphic reconstruction from transverse sections. The fore gut is separated from the entoderm of the blastodise and is approximately one-sixth the length of the body. The lateral walls of the mid and hind gut are still sepa- rated at their bases almost throughout by a considerable ventral cleft, and meet only a few sections anterior to the tail to form the lower and hinder walls of the neurenteric canal. The first gill pouch is a shallow depression, present on one side only. The second gill pouch is indicated by a very slight depression in the dorsal part of the lateral wall of the pharynx immediately pos- DEVELOPMENT OF THE ELASMOBRANCH LIVER 309 terior to the first pouch. Its ventral portion is not as yet evaginated. As the fore gut approaches its connection with the blasto- dermic entoderm it is at first broadly ovoid in cross section with the narrower end of the oval upward and its vertical diameter increases posteriorly. Back of the fore gut the archenteron is flattened transversely until it is little more than a high, narrow fold of entoderm, the transverse diameter of which is less than one-fourth of the vertical diameter. The lateral walls of the gut for a short distance behind and also a little in front of the point of union of the fore gut and the mid gut are differentiated into dorsal and ventral zones. The epithelium of the dorsal zone is Fig. 1 Lateral view of the archenteron of anembryo of 19 to 20 segments, 4.0 mm. in length (H.E.C. 930). X 20. F.g., fore gut; G.p., gill pouches; Hep.a., hepatic area. from 25 to 30.» in thickness and contains two or more rows of more or less interlocking oval nuclei. This is the primitive con- dition found throughout the walls of the archenteron, both dor- sally and ventrally in earlier stages. Close to the lower border of the dorsal zone there is on either side a shallow and not always continuous longitudinal groove which later becomes a definite and important landmark. For these I suggest the name para- archenteric grooves. The ventral zone in this region, as is seen from following its later history, represents the liver anlage. Here the epithelium is approximately half as thick as that of the dorsal zone, and the nuclei, which form a single row only, lie in the basal portion of the epithelium. . Ordinary stains do not bring out definite cell walls in either the dorsal or ventral zones at this stage. There 340 RICHARD E. SCAMMON are no definite boundaries, except the dorsal one to the hepatic area at this time. The arrangement of nuclei just described extends ventrally nearly to the point where the lateral walls of the archenteron turn laterad as a part of the blastoderm. Longi- tudinally the hepatic region extends forward a little past the posterior-end of the fore gut to become indistinguishable in the general ventral enlargement of the pharynx already referred to. Its characteristics are less noticeable as we follow the gut poste- riorly and 100 « behind the point of union of fore and mid gut the hepatic area is indistinguishable from the other entoderm. There is but a slight indication of evagination of the liver area. Al- though the lumen between the walls of the ventral zone is nearly twice as wide as that above, this width is due mainly to the decrease in the thickness of the walls themselves, the entire trans- verse diameter of the gut being a little greater dorsally than ventrally. : A slightly older embryo having 24 trunk segments and 3.6 mm. in length, which is a little more advanced than the Normal plate No. 20 (Seammon 711), gives a clearer picture of the liver anlage. The pharynx, from which two well formed gill pouches pro- ject and fuse with the skin ectoderm, is followed by a short seg- ment of gut which represents both the oesophagus and the ante- rior part of the stomach. This segment is elongately oval in cross section with very much thickened lateral walls. It is somewhat produced ventrally as it approaches the anterior wall of the yolk- stalk, and in this ventral region shows lateral expansion. The archenteron extends forward forming a large anterior recess above the yolk in front of the anterior wall of the yolk-stalk. Immedi- ately behind the point of union of the fore gut with the yolk- stalk the archenteron has the same form as in the younger embryo, being greatly elevated and flattened transversely. The distinction between dorsal and ventral zones is fairly marked, and the para-archenteric grooves can be traced along the gut above the hepatic region, although in places they are very faint and shallow. The ventral zone in this region is now distinctly curved outward, forming a pair of shallow, lateral diverticula which extend approximately 100.4 posterior to the anterior vitello- DEVELOPMENT OF THE ELASMOBRANCH LIVER 341 intestinal junction. The walls of these lateral diverticula are of the same thickness as those of the dorsal zone, i.e., 25 to 30 u, but the nuclei are somewhat more elongated and lie at the basal ends of the cells, leaving a clear zone towards the lumen of the gut. The boundaries of the cells are faintly distinguishable. fe RS) ‘af Fig. 2 Transverse section of an embryo of 19 to 20 segments, 3.0 mm. in length (K.U.E.C. 451). 0.06 mm. posterior to the anterior wall of the yolk stalk. > 100. Hep.d., hepatic diverticulum; P-arch.g., para-archenteric groove. Fig. 3. Tranverse section of an embryo of twenty-four segments, 3.6 mm. in length (S.C. 14). 0.09 mm. posterior to the anterior wall of the yolk stalk. X 100. Hep.d., hepatic diverticulum; P-arch.g., para-archenteric groove. Above and below the diverticula the nuclei are broadly oval and scattered through the thickness of the epithelium, and there are no distinct cell outlines to be seen with ordinary stains. Figure 3 is a transverse section of this embryo 90 u behind the anterior wall of the yolk-stalk. 342 / RICHARD E. SCAMMON A distinct step in development is shown by an embryo 5 mm. in length (K.U.E.C. 449%). This embryo has segments and three gill pouches, none of which open to the exterior. The gut is still connected with the yolk entoderm by a yolk-stalk, which has an antero-posterior diameter equal to nearly half the length of the archenteron. A lateral view of the liver region is outlined in figure 4, which is a graphic reconstruction, with the hepatic area outlined with broken lines. The lateral hepatic diverticula are somewhat better defined, both dorsally and ventrally, than in the preceding stage and are somewhat deeper. They show in a more marked way the histologic differentiation described for the preceding embryo, as is illustrated by figures 5 and 6, for the cells have lengthened so rapidly that epithelium is one-fourth to one-half thicker than that of the dorsal zone of the gut above it. Numerous mitoses indicate the rapid growth now taking place in this region. It will be noticed from figure 4 that the lateral liver diverticula now extend forward distinctly beyond the ante- rior wall of the yolk-stalk. Here the right and left pouches are fused, forming a single median and ventral pouch in the posterior part of the fore gut. The finer structure of this anterior part is illustrated in figure 5. It shows the same character and dis- tinction from the remainder of the gut wall as does the posterior part of the anlage. The right and left omphalo-mesenteric veins are now present, although of small caliber. A slightly older embryo 6.4 mm. long shows somewhat the same stage of development and the liver region has been reconstructed in wax. Figures 30 and 31 show anterior and right lateral views of this object. The entire embryo is inclined markedly to the left. The fore gut, ovoid in cross section, becomes immediately flattened and triangular after passing the anterior wall of the yolk- stalk.. The lateral hepatic diverticula are distinctly outlined and fuse together, forming anteriorly a ventral pouch in the floor of the fore gut. The antero-posterior length of the hepatic anlage 3 The following designations of embryos are employed in this paper: H.E.C. = Harvard Embryological Collections. K.U.E.C. = Kansas University Embryo- logical Collection. $.C. = Author’s Collection. Rs PR arch. 9: M.hep.p. Fig.4 Graphic reconstruction of a portion of the fore and mid gut of an embryo 5mm. long (K.U.E.C. 449). X50. The dotted lines indicate the borders of the hepatic thickening. Lines A and B indicate the planes of sections represented in figures 5 and 6. F.g., fore gut; Hep.d., hepatic diverticula. Fig.5 Transverse section of the same embryo 0.03 mm. in front of the anterior wall of the yolk stalk. > 100. Fig.6 Transverse section of the same embryo 0.06 mm. posterior to the anterior wall of the yolk stalk. x 100. Hep.d., hepatic diverticula, M.hep.p., anterio- median hepatic pouch, formed by the fusion of the lateral hepatic diverticula. P-arch.g., para-archenteric grooves. 343 344 RICHARD E. SCAMMON is 0.30 mm. The length of the fused anterior parts of the lateral diverticula is 0.13 mm., while that of the still separated posterior portions is approximately 0.17 mm. The posterior part of the lateral liver anlagen, however, can hardly be called diverticula, as they are little more than thickened plates of cells. The histo- logie differences between the liver diverticula and the remainder Fig. 7 Transverse section of an Acanthias embryo 6.4 mm. long (S.C. 19), 0.05 mm. posterior to the anterior wall of the yolk stalk. X 100. Hep.d., hepatic diverticula; P-arch.g., para-archenteric groove. of the archenteron walls are shown in figure 7. The nuclei in the walls of the lateral diverticula are no longer arranged in a single layer, but are irregularly placed in the basal halves of the. cells. Their elongation is noticeable. The cytoplasm of the cells of the hepatic area is condensed and stains darkly as compared with that of the cells above. The para-archenteric grooves are very DEVELOPMENT OF THE ELASMOBRANCH LIVER 345 broad and shallow and can be followed with difficulty above the lateral diverticula. Two marked changes now take place in the liver anlage, bring- ing about the condition described by Brachet (’96) and others as the primitive one. These are the appearance of the gall bladder and the distinct lateral and dorsal evagination of a part of the lateral hepatic diverticula. An embryo 7 mm. in length (H.E.C. 752), which is probably a little younger than No. 21 of the Normal plate series and a little older than Balfour’s Stage H is the earliest specimen in which I have observed any indication of the gall bladder. Both the separated posterior parts and the anterior median pouch, formed by the fusion of the anterior ends of the lateral hepatic diverticula, are more pronounced than in the em- bryo just described. The anterior wall of the yolk-stalk is much thickened over its entire extent, but particularly just below the point where it becomes continuous with the floor of the fore gut. The gall bladder is represented by a very shallow median depres- sion at this place. Numerous mitoses indicate that the epithe- lium is growing rapidly in this region. The early evagination of the gall bladder must take place with some rapidity, as it is very difficult to find any specimens between the stages when this structure is entirely absent and when it is a deep, well-marked pouch. A specimen which illustrates both the early development of the gall bladder and the growth of the lateral diverticula, and is quite comparable with the first members of Hammar’s (’93) and of Brachet’s (’96) series of models, has been reconstructed and figures 33 and 34 are anterior and left lateral views of the model. This specimen, which is 7.5 mm. in length (H.E.C. 1503 and No. 24 of the Normal plate series), has 54 to 55 trunk segments and four gill pouches, two of which open exteriorly. The spiral valve makes one and one-third turns of the gut. The distance from the last (fourth) gill pouch to the anterior wall of the yolk-stalk is approximately one-fourth of the complete length of the alimentary canal and about equal to the antero-posterior diameter of the yolk-stalk. The lateral hepatic diverticula are differentiated into three parts. Anteriorly they are fused, form- 346 RICHARD E. SCAMMON ing the single median pouch already mentioned which now pro- jects downward from the floor of the fore gut. Continuous with the median pouch thus formed are the middle parts of the diver- ticula which are expanded laterally and dorsally and which will be referred to hereafter as the lateral hepatic pouches. The lateral hepatic pouches extend backward as far as the anterior wall of the yolk-stalk, and there become continuous with the posterior parts of the lateral diverticula which remain almost unchanged from their slightly expanded condition of earlier stages, and which will he referred to as the pars ductus, as it is from them that the major portion of the ductus choledochus is formed. The left lateral pouch becomes continuous with the pars ductus of that side without any distinct line of demarcation. On the right side, however, the pouch ends abruptly by projecting nearly at right angles from the gut wall. Deep grooves, of which the left is the more pronounced, intervene between the latter walls of the ventral part of the fore gut and the mesial walls of the dor- sally growing lateral pouches. These indicate the beginning of the process by which the liver will be eventually cut off from the gut tube above. The gall bladder is present as a deep ventral pouch, lying between the median liver pouch in front and the , anterior wall of the yolk-stalk behind. Its walls are directly continuous with the ventral part of the liver pouch and the pars ductus above, but a slight longitudinal groove marks the bound- ary between the structures. This groove becomes deeper as it proceeds anteriorly until a point is reached about one-fifth of the length of the pouch from its anterior wall. Here the groove is entirely absent and there is thus left.a small anterior expanded segment of the gall bladder stalk which is the anlage of the primi- tive cystic duct. A point worth emphasis is that the entire liver anlage shows a slight rotation to the left and that the left lateral liver pouch shows a greater dorsal growth than does the right. The para-archenteric grooves have remained unchanged. The changes which now follow are those of passive growth. The lateral pouches expand transversely and become almost globose in outline. At the same time there is a slight growth DEVELOPMENT OF THE ELASMOBRANCH LIVER 347 dorsally which deepens the groove between them and the gut mesially. The pars ductus expands somewhat and becomes more sharply marked off from the archenteron posteriorly. The anterior median pouch also shares in this expansion but shows no other changes. Likewise the gall bladder becomes rotund, a distinct groove intervenes between its dorsal-anterior angle and the liver anlage and a ventral notch of some depth separates the sack from the anterior wall of the yolk-stalk behind. In front of this ventral notch the sac is still continuous with the liver anlage proper, but the longitudinal construction between the two structures mentioned in the description of the preceding embryo is present in a more distinct form. These changes are illustrated in figures 35 and 36, of a wax reconstruction from an embryo 9 mm. in length, the general anatomy of which has been previously illustrated in graphic reconstruction in figure 11 of the Normal plates of Acanthias. A little later, as shown in an embryo of sixty somites with three open and two closed gill pouches and two complete turns of the spiral valve, the lateral pouches lose their expanded outline, and becoming flattened laterally, enter upon a decided dorsal growth (figs. 37 and 38). At the same time their posterior margins become sharply differentiated so that they extend out from the gut at an abrupt angle and their distal edges show several slight irregularities. The anterior median pouch remains practically unchanged. The lateral grooves along which the liver eventually separates from the fore gut above it are now considerably deep- ened and extends the entire length of the line of attachment of the liver evagination, although they are still shallow anteriorly. The left lateral pouch bears on its lateral surface three small longitudinal ridges. These together with the dorsal irregulari- ties mentioned above constitute the anlagen of hepatic tubules and will be discussed in the section dealing with these structures. The gall bladder while no larger than in the preceding embryo is separated from the anterior wall of the yolk-stalk by a deeper ventral notch and the constriction between the sack and the median part. of the liver above is more pronounced. Both this 348 RICHARD E. SCAMMON and the preceding stage show a slight but distinct rotation of the anterior part of the liver to the left around the fore gut as an axis. A considerable advance in development is seen in an embryo only a millimeter longer than the preceding one. This specimen corresponds fairly well with Balfour’s stage I, or No. 24 of the Normal plate series the embryos of which measured 11.5 mm. It has sixty-five segments, three open gill slits and two unopened gill pouches and four turns of the spiral valve. A reconstruction of the liver and adjoining archenteron is illustrated in figures 28, tow ~ Sys S as Sin wns Y if j J Sal ) EY c i ‘i > duct. lat. Fig. 8 Three transverse sections through the liver region of an Acanthias embryo 10mm. long (S.C. 20). X 50. A, Through the anterior part of themedian hepatic pouch (pars hepatica mediana). B, Through the posterior part of the median pouch (pars ductus mediana). C, Through the gut just posterior to the liver proper showing the pars ductus lateralis. F.g., fore gut; G.bl., gall bladder; Lat.hep.p., lateral hepatic pouch; P-arch.g., para-archenteric groove; P.duct.lat., pars ductus lateralis; P., pars hepatica mediana; V.omph.l., V.omph.r., left and right omphalo-mesenteric veins; X, anlage of the ductus choledochus. 39 and 40. The process by which the hepatic pouch will even- tually be separated from the gut above is well under way. The median hepatic pouch from which the lateral pouches spring and to which the lateral pouches are attached is rather triangular in cross section anterior to the origin of the lateral pouches. The broader part is below and the narrow dorsal extremity joins the floor of the fore gut anteriorly a little to the right of the median line (fig. 8 A). The ventral part of the gut is also rotated to the right so that | these two structures join at an oblique angle thus forming a broad * ‘ DEVELOPMENT OF THE ELASMOBRANCH LIVER 349 shallow groove on the left hand side while on the right they form a smooth somewhat convex surface. Posteriorly the median hepatic pouch is continuous with a short segment of the gut which in turn becomes attached to the yolk stalk. The para-archen- teric grooves are still distinguishable and mark the plane of union between the hepatic anlage and the gut (fig. 8, A, B and C,-P- arch.g.). Two parts can be distinguished in the median pouch, an anterior one which projects a little in front of the anterior end of the attachment of the lateral pouches, and a posterior part which is directly continuous with the former and from which the lateral pouches and the gall bladder take origin. Although these divi- sions are not sharply marked off at present, they later become quite distinct. The anterior one from its later history may be called the pars hepatica mediana because it shares with the lateral pouches or pars hepatica lateralis in the formation of hepatic ducts and trabeculae. The posterior later develops.into a part of the ductus choledochus and may be called the pars ductus mediana in distinction to the anterior and to the pars ductus lateralis formed from the posterior portions of the original hepatic diverticula. The upper surface of the posterior part or pars ductus mediana of the median pouch lying on either side of this dorsal connection with the gut already shows a peculiar modeling indicative of the course which will be eventually taken by the ductus choledochus (fig. 28). There is a marked expansion which extends from the right anterior angle of the median pouch obliquely backward to the posterior left corner. The anterior, 1e., right portion of this swelling is the more marked. From the posterior edge of the middle pouch this expansion is continued backward into the mid gut as a symmetrical lateral expansion of the ventral part of the ‘connecting piece’ between the hepatic and stomach anlagen above it to the yolk-stalk and overlying gut. As seen from their position these lateral expansions are the remains of the posterior ends of lateral hepatic diverticula or | pars ductus lateralis (fig. 8, C). Thus there can already be recog- nized two distinct parts of the ductus choledochus: an anterior asymmetric portion and a posterior part which is symmetrically placed. THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO. 3 350 RICHARD E. SCAMMON The lateral pouches have continued their dorsal growth and now extend as far upward as the dorsal surface of the gut. Their upper parts are expanded, particularly posteriorly, so that a proximal constricted stalk and a distal expanded portion can be distinguished. In earlier stages as shown by figures 33 and 37, the origin of each lateral pouch was continuous with the entire lateral edge of the median one, but at this stage it is confined to the posterior four-fifths of this edge. The dorsal part of each lateral pouch is curved a little medially. All the external lateral surface of each lateral pouch is corrugated with rather irregular longitudinal ridges which are somewhat broken by shallow trans- verse fissures (fig. 40). Similar ridges are forming on the ventral surface of the median pouch anterior to the gallbladder. The dorsal edge of each median pouch is also rendered exceedingly irregular by the several small pouches springing from it. All of these structures are the anlagen of hepatic tubules the forma- tion of which was referred to in the description of the preceding embryo. The gall bladder is now a large thick-walled sae, ovoid in shape and somewhat flattened dorso-ventrally. Distinct grooves sepa- rate it from the median liver pouch and connecting piece behind and along its dorsal edge. These grooves are however deeper posteriorly than anteriorly as might be expected from their his- tory in earlier stages. The extreme anterior tip of the gall blad- der is drawn to a point and projects very slightly forward below the anterior part of the median liver pouch. IV. CONCLUSIONS All the main divisions of the liver are now established and before giving an account of their later history it may be well to sum- marize the development of the organ up to this stage. The semi- diagrammatic models shown in figures 9 to 12, illustrate this process. They are of embryos 3.6, 6.4, 7.5 and 9 mm. long respectively, and are based upon wax reconstructions and meas- urements of specimens described in the preceding pages. In each a portion of the mid and fore gut is represented as resting on a block of yolk. The dorsal half of the archenteron is cut away DEVELOPMENT OF THE ELASMOBRANCH LIVER 351 so that one looks down on the interior of the ventral half of the gut from above and a little behind. Figure 9 shows the liver as a pair of shallow lateral diverticula, lying mainly behind the point of union of fore gut and blastodermic entoderm or the anterior wall of the yolk-stalk. As the embryo is elevated and farther separated from the blastoderm the anterior wall of the yolk- stalk retreats posteriorly. This brings the anterior ends of the lateral diverticula in contact and they fuse more and more, form- ing the median liver pouch and producing the condition shown in figure 10. Here the liver anlage is U-shaped with the limbs of the U turned posteriorly and slightly divergent. The fusion of the lateral diver- ticula is continued along with the posterior progression of the anterior wall of the yolk-stalk. At the same time parts of these structures undergo unequal growth. In each diverticulum the middle part above and a little behind the fused median portion begins a rapid dorsal and lateral growth, producing the structures known variously as ‘lateral pouches’ ‘ébauche hépatique,’ and ‘Seitendivertikel.’ The posterior parts of the primitive lateral diverticula, or pars ductus, which extend backward to or beyond the anterior wall of the yolk-stalk share but little in this growth, but remain unchanged in their primitive condition as a pair of shallow lateral diverticula until at a much later period, they are transformed into a part of the ductus choledochus. In the meantime the gall bladder arises as an out-pouching of the dorsal part of the anterior wall of the yolk-stalk and being somewhat cut off from that structure by the posterior growth of the fore gut comes to lie between it and the median liver pouch anteriorly and with the lateral median pouches bounding its sides. This stage is represented in figure 11. Figure 12 shows a some- what later stage modified from the preceding by the greater expan- sion of all parts of the hepatic structure and by a still greater elongation of the fore gut. This account of the development of the liver is to some extent in accord with that given by Hammar (’93) as opposed to the idea of a single median ventral anlage as advanced by Balfour (76), Laguesse (’93), Brachet (96) and Choronschitzky (00). Sp2 RICHARD E. SCAMMON g Z Z gZ Z Z Z Z Figs. 9, 10, 11,12 A series of semi-schematic reconstructions to illustrate the early development of the liver; all X 50. The plan of reconstruction is explained in the text on p.350. F.g., fore gut; G.bl., gall bladder; Lat.hep.p., lateral hepatic pouches; Med.hep.p., median hepatic pouch; Hep.d., hepatic diverticula; P.duct. lat., pars ductus lateralis; Sp.v., spiral valve; Y.w., anterior wall of the yolk stalk. Fig. 9 Embryo 3.6 mm. long. ~° Fig. 10 Embryo 6.4 mm. long. Fig. 11 Embryo 9mm. long. DEVELOPMENT OF THE ELASMOBRANCH LIVER 353 From a short study of early Torpedo embryos I am inclined to think that in this form the stage in which the liver exists as a pair of lateral diverticula must be very brief -if at all present, because embryos of this form separate from the blastoderm at an earlier period than do Acanthias embryos and have a compara- tively small yolk-stalk. The rapid formation and elongation of the fore gut accompanying these changes may involve the hepatic areas before they are differentiated as pouches. As regards Acan- thias and probably other Selachii, it appears to me very probable that investigators have been misled from a study of embryos which have advanced to a considerable extent in the process of development. It is interesting to note that in embryos of other groups of animals possessing large yolk-laden ova the liver forms at a stage when other organs are in quite a primitive condition, and in only slightly teleolecithal ova the time of origin is still younger. At the time when Balfour, Brachet and Laguesse record the appearance of the liver in elasmobranch fishes the embryo is well established, several gill slits are fully formed, the sense organs are completely invaginated, the spinal nerve anlagen are laid down and the limb fundaments are about to.appear. The earliest anlage of the liver is not extensive and can hardly be recognized - without a previous study of somewhat later stages. Again after the primitive paired liver diverticula are well formed they are often to a considerable extent obliterated by the falling of the embryo to one or the other side as it becomes top-heavy by separa- tion from the yolk which has supported it up to this time. This flexure causes one or more large transitory folds which tend to render inconspicuous the pouch in the side upon which the embryo’ comes to lie, and at the same time almost obliterates the opposite pouch by stretching. The histologic characters of these areas © remain unchanged however. Figure 13 is a cross section through the liver region of an embryo 4.8 mm. in length, showing these changes. Although in this form of selachian at least the anlage of the liver is a paired one, it does not follow that this is the original condition of the structure. It seems probable that it has been brought about purely by the mechanical influence of the large 354 RICHARD E. SCAMMON amount of yolk present by which the original tube of entoderm is spread out plate-like upon an almost flat surface of the yolk sub- stance. In such a case the ventral portion of the original tube would form the peripheral portion of each lateral half of the plate and the folding of the plate into a tube again in the course of _ Fig.13 Transverse section of an Acanthias embryo 4.8 mm. long (H.E.C. 1398), showing the effect of the inclination of the embryo upon the lateral hepatic diverticula. x 50. Hep.d., hepatic diverticula. the separation of the embryo from the yolk would approximate once more the two separated parts. These lateral diverticula may then be regarded simply as the potential halves of an original ventral pouch which begin their expansion before their union along the ventral median line takes place. As might be expected DEVELOPMENT OF THE ELASMOBRANCH LIVER 355 this union does not occur at the same time along the entire antero- posterior length of the liver but proceeds from the cephalic end backward, thus producing the U-shaped figure shown in figures 9 and 10. It appears that too much importance has been placed upon the position of the liver anlagen in regard to the anterior intestinal portal. Laguesse (’93), Brachet (’96) and Choronschitzky (’00) all emphasize this point. This structure, however, is constantly shifting posteriorly as has been demonstrated by Mayr (’97), and the location of the liver in front of it holds good only for stages which are well advanced. The location of the liver as immedi- ately behind the sinus venosus is perhaps of more value, but in Acanthias at least, the liver is present before the two omphalo- mesenteric veins become confluent to form the endothelial heart, or indeed before they are represented by definite endothelial tubes. Laguesse has already called attention to the fact that in Acan- thias the gall bladder appears somewhat later than the remainder of the hepatic apparatus, and seems to be developed from the anterior wall of the yolk-stalk rather than from the posterior part of the median liver pouch. Hammar (’93, ’97) appears to hold the same opinion in regard to Torpedo. I believe that my _ sections and models bear out this conclusion and that this struc- ture can be properly interpreted as an organ arising quite separate from the hepatic anlage at the juncture of the pars ductus of the lateral liver diverticula and the floor of the gut, as represented by the anterior wall of the yolk-stalk portal. The shifting of the sac anteriorly so that its duct comes to lie in front of the openings of the hepatic ducts into the ductus choledochus will be discussed in the following section. All specimens after the stage when the median and lateral hepatic pouches are formed show a small but constant rotation of the hepatic anlage to the right. This rotation seems without doubt to be a part of that greater one which produces the spiral valve. Like the latter it is from the left to the right side, ie., clockwise around an axis corresponding to the longitudinal axis of the gut, and it is coincident with it, appearing when the embryo has from 50 to 60 segments and has reached a length of 6 to 7 mm. 356 RICHARD E. SCAMMON Its extent is but slight as compared with that of the posterior portion of the gut, being at most not over 15 degrees. The hinder and lower portion of the hepatic anlage is less affected by this twisting than is the anterior free part presumably because its attachment to the vitelline duct is still considerable in extent and must offer some resistance to whatever force it may be that pro- duces the rotation. That this portion of the alimentary tract is affected to a slight degree however is shown by the broad shallow groove which appears in the posterior half of the left wall of the yolk-stalk and which has been figured and described in the Normal plates (Secammon ’11), under the term ‘Lateral erooyve of the vitelline duct,’ and which may be seen in the figures of reconstructions of embryos 7.5, 9 and 11.5 mm. in length respectively, in that paper. The gut anterior to the yolk-stalk also shows some rotation, being twisted to the right as is indicated by the angle formed by its lumen with the mid-sagittal plane of the body. PAR Dae I. DESCRIPTION OF FULLY FORMED BILIARY APPARATUS Before attempting to describe the development of the gall - bladder and liver ducts, it may be well to outline the form of these structures in the late embryo or new-born fish and to pre- sent the terminology which will be employed in the remainder of this paper. In large embryos and new-born specimens of Acanthias the liver is a large viscus occupying nearly half of the abdominal cavity. It consists of two lateral lobes which are united ante- riorly by a median mass which stretches completely across the body cavity posterior to the septum transversum. From the right ventral and posterior margin of the median mass a small pointed process extends backward and to the left. As the gall bladder is imbedded in this mass it has been termed the cystic lobe. The cystic lobe lies directly ventral to the stomach and to the left of the cephalic end of the large internal yolk sac. | . | DEVELOPMENT OF THE ELASMOBRANCH LIVER an The gall bladder is an elongated tubular sae which lies along the right margin of the cystic lobe. It is imbedded in hepatic parenchyma except for a little of the right surface which receives a peritoneal investment. The cystic duct arises from the anterior end of the gall bladder and proceeds directly dorsally. It then Fig. 14 A dissection of an Acanthias embryo 20 cm. instlength. x 2. The ventral abdominal wall has been cut away and the vitelline duct severed at its connection with the internal yolk stalk. The gall bladder ahd main hepatic ducts have been dissected out. The large veins of the liver have been omitted from this drawing. C.l., cystic lobe; D.chol., ductus choledochus; D.cyst., cystic duct; D.hep.l., left hepatic duct; D.vit., vitelline duct; G.bl., gall bladder; J.y.s., internal yolk sac; L.l., lateral lobe; Panc., pancreas; V.int., valvular intestine; V. subint,, subintestinal vein. Fig. 15 Diagrammatic representation of the gall bladder and liver ducts of Acanthias as seen from above. D.chol., ductus choledochus; D.cyst., cystic duct; D.hep.l., left hepatic duct; D.hep.r., right hepatic duct; G.bl., gall bladder; R.g.l., anterior left hepatic ramus; R.a.r., anterior right hepatic ramus; R.l.m., left medial hepatic ramus; R.p.l., posterior left hepatic ramus; R.p.r., posterior right hepatic ramus; R.r.m., right medial hepatic ramus. makes a sharp semicircular curve and proceeds posteriorly to join the ductus choledochus. In older embryos and adults there is no line of demarcation between the cystic and common bile ducts. The ductus choledochus extends backward ventral to the ret yes a wh eal i ae | % & 358 RICHARD E. SCAMMON internal yolk sae joining the valvular intestine on the left side of the first turn of the spiral valve. Figure 15 shows the gall bladder and hepatic ducts in diagram. The main right and left hepatic ducts join with the ductus chole- dochus obliquely, the left gaining entrance in front of the right. The distance between the ostia of the two ducts varies in different specimens. The left duct after extending a short distance an- teriorly arches far out laterally and there turning backward passes posteriorly in the left lateral lobe. The right duct makes a sharp arch anteriorly and then passes backward into the right lobe. The main lateral hepatic ducts give rise to numerous small hepatic tubules and to several larger rami. The former are extremely irregular in form, origin and number, but the latter, although displaying great variation in position can in most cases be reduced to the following classification: (1) right medial hepatic ramus, (2) left medial hepatic ramus, (3) anterior right hepatic ramus, (4) posterior right dorsal hepatic ramus, (5) anterior left hepatic ramus, (6) posterior left dorsal hepatic ramus. The right medial hepatic ramus varies considerably in the place of origin, commonly it is attached to the right duct near its proxi- mal end. The left medial hepatic ramus commonly takes origin from the proximal part of the left hepatic duct but may in some cases be attached to the ductus choledochus or even to the base of the right hepatic duct. The anterior right and the anterior left rami generally arise from the summit of the anterior arch formed by each of the main hepatic ducts, but the left ramus may attach either to the ductus choledochus, or as I have observed in one embryo, to the base of the main right hepatic duct. The posterior dorsal hepatic rami arise from the hepatic ducts either at the lateral extremity of the anterior arch or in the anterior part of their posterior course. They seem to be fairly constant in position. Minor variants from the above scheme are common and simple rami differ much in size or may be replaced by two or more smaller ones. The terminology used here is based upon the development of these structures as will now be described. DEVELOPMENT OF THE ELASMOBRANCH LIVER 359 II. DEVELOPMENT OF THE HEPATIC DUCTS AND THEIR RAMI The elements entering into the formation of the hepatic ducts are the anterior portion of the median liver pouch or pars hepatica mediana, and the right and left lateral hepatic pouches or the pars hepatica lateralis.t These structures are converted into the main hepatic ducts found in the fully developed embryo by means of reduction in caliber both relative and actual, by elongation, and by partial separation from the posterior portion of the median liver pouch or pars ductus mediana. That such processes take place has been recognized by Balfour (76), Hammar (’93), Brachet (’96), and other investigators. The details have not been described. The minor ducts are formed in Acanthias in much the same manner as are the major ones, by the differentiation and elongation of the proximal parts or pedicles of certain fairly defi- nitely placed groups of tubules which arise from the surface of the embryonic structures which form the main ducts. This method of formation of the minor ducts probably holds only for selachians in which the omphalo-mesenteric veins are compara- tively small and develop at a late stage. An account of the development of these ducts may begin with the description of an embryo 15 mm. in length (H.E.C. 227 and No. 26 of the Normal plate series) the general anatomy of which is illustrated in figure 13 of the Normal plates of Squalus acanthias. The main divisions of the liver of this specimen and the proximal part of the hepatic tubules arising from them have been recon- structed and figures 41, 42 and 44 are right lateral, left lateral and anterior views of this object. ‘The median hepatic pouch is completely separated from the gut above and this separation has extended backward convert- ‘The use of this and the following terms of this paragraph is somewhat of a departure from the classification of the components of the selachian liver pouch into ‘pars cystica’ and ‘pars hepatica’ as proposed by Brachet (’96, 797) on the basis of a similar classification employed by Goeppert (’93) in his description of the development of the liver in Teleosts. The term ‘pars cystica’ as used by Brachet includes the ‘pars ductus mediana’ and ‘pars ductus lateralis’ as employed here, as well as the anlage of the gall-bladder and cystic duct. If the conception as presented here, of the gall bladder as an organ with an origin distinct from the liver, is a correct one, then this term ‘pars cystica’ can hardly be properly applied 360 RICHARD E. SCAMMON ing the pars ductus lateralis into a short tube of large caliber. This is the middle part of the ductus choledochus. It joins with the floor of the duodenum a little to the right, thus preserving the same relation observed in younger embryos. The obliquely placed swelling upon the dorsal surface of the median pouch which represents the course of the distal portion of the ductus choledochus is present but is not so marked as in the embryo 10 mim. in length described in the preceding section (p. 348). The pars hepatica mediana or anterior part of the median pouch is broadly continuous with the pars ductus medialis behind and with the lateral pouches posteriorly and laterally. Its anterior surface (fig. 44) is rendered extremely irregular by the formation of a number of hepatic tubules. The origin of these structures from ridges in the pars hepatica was noted in connection with the description of an embryo 10 mm. in length, in the preceding section of this paper. At the present stage the tubules arising from the pars hepatica mediana are little more than short conical evaginations of the pouch wall and only one shows any evidence of the complex branching which all soon undergo. The tubules of the pars hepatica mediana are divided into two groups, a right and a left, by a deep vertical furrow which lies somewhat to the left of the median plane and extends from the dorsal to the ventral surface of the pouch.. The right subdivision thus formed is more extensive than the left, but a smaller number of tubules arise from it. The groups of tubules established by this subdivision will be termed in this paper the right medial group and the left me- dial group respectively. The lower surface of the pars mediana remains smooth at this stage and rests upon a mass of mesenchyma which extends from the anterior surface of the gall bladder to the anterior mesothelial wall of the liver. to all of these structures and to use it for structures which are later wholly incor- porated in the ductus choledochus and not in the vessica fellae or its duct at all, seems inadvisable. The use of the expression ‘mediana’ in connection with ‘pars ductus’ is not intended to convey the meaning that this portion of the ductus choledochus is a direct derivative of the median part of the gut primarily, but that it is formed from the median pouch produced by the fusion of the anterior parts of the original lateral diverticula while the more posterior part of the ductus chole- dochus is formed from the hinder parts of the lateral diverticula without the inter- vention of a median pouch stage. DEVELOPMENT OF THE ELASMOBRANCH LIVER 361 The lateral pouches merge into the pars hepatica mediana anteriorly and the distinction between these parts in this region is only possible through an examination of the tubule formation. Posteriorly, however, the proximal portion or stalk of each potich is constricted and elongated to form the hinder part of a broad short duct connecting the pars ductus of the median pouch with the distal expanded portions of the lateral ones. This condition is illustrated by the transverse section shown in figure 16. © pice ea | =o ke Poe) Fig. 16 Transyerse section through the liver of an Acanthias embryo 15 mm. long (H.E.C. 227). Xx 125. G.bl., gall bladder; L.hep.p., left hepatic pouch. P.duct.med., pars ductus mediana; R.hep.p., right hepatie pouch. 362 RICHARD E. SCAMMON The mesial surfaces of the lateral pouches are smooth except for some minor fissures and the gutter like spaces between them and the pars mediana are occupied by mesenchyma and the vitel- line veins. The lateral surface of each pouch is almost obscured by the numerous hepatic tubules which arise from it. The dorsal growth of the pouches so evident in early stages has now come to an end and their dorsal margins hardly extend above the ventral surface of the stomach as is seen in figures 41 and 42. A number of large trunk-tubules arise from them. As in the pars hepatica mediana all the tubules of the lateral pouches tend to gather in certain fairly well defined groups. These consist, on either side, of an anterior and a posterior group, and the latter is less definitely subdivided into a dorsal and a ventral cluster. The tubules of the anterior groups spring from the dorsal half of the anterior part of the lateral pouch leaving a ventral area below which is smooth or occupied only by small tubules in the process of forma- tion. The posterior group is much larger and its two subdivi- sions occupy the entire posterior half and hinder margin of the pouch. As will be seen from figure 41, these groups are not com- pletely separated, as small and less developed tubules intervene in some places. The formation of these minor tubules continues until a much later period. The tubule groups of the anterior part of the left lateral pouch and of the left side of the pars hepatica mediana lie much closer together than those of the opposite side. This early arrangement of the hepatic tubules into groups is of much importance for, as has been stated, while the main hepatic ducts are produced by the elongation and narrowing of the caliber of the lateral pouches, each group of tubules becomes iso- lated by the formation of a common stalk which later develops into one or more rami of the minor hepatic ducts. The arrange- ment of the tubule groups is expressed in tabular form in table 1. A reconstruction, illustrated in figures 43, 45 and 46 of an em- bryo but 0.5 mm. longer than the preceding but which resembles in general anatomy the average embryo of 18 mm., shows more clearly the process of duct formation. 'The common bile duct is now three times as long as its greatest diameter and the pars duc- DEVELOPMENT OF THE ELASMOBRANCH LIVER 363 tus mediana has lost somewhat of its sac-like form and appears as an irregular dilated chamber which is broader in front than behind and receives the broad short cystic duct from below and the proxi- mal parts of the lateral pouches from the sides. The pars hepatica mediana is somewhat elongated and is still broadly continuous with the pars ductus posteriorly. The formation of tubules from it has progressed considerably and now involves the ventral as well as the anterior surface. The division of this part of the, median pouch into right and left segments by a vertical fissure is well marked. This fissure lies in the same sagittal plane as the left lateral wall of the pars ductus behind it, thus showing a marked TABLE 1 Arrangement of hepatic tubule groups Pars hepatica mediana oo — Right medial Left medial tubule tubule group group Right hepatic pouch Left hepatic pouch Anterior right Posterior right Anterior left Posterior left tubule group tubule group tubule group tubule group Dorsal Ventral Dorsal Ventral cluster cluster cluster cluster shift to the left. On the right side the pars hepatica mediana is no longer confluent with the anterior part of the lateral pouch but is separated from it by narrow zone of tubule free surface (fig. 46). On the left side however the pars hepatica mediana extends far laterally and is continuous with the left lateral pouch posteriorly. The lateral pouches also show several changes. Their proximal stalks are elongated and the size of their distal expansions is much reduced. The connecting stalk of the left pouch with the pars ductus mediana is shifted so far forward that its posterior margin lies in the same transverse plane as the anterior margin of its fellow of the opposite side. The distal part of the left pouch is 364 RICHARD E. SCAMMON also farther separated from the posterior part of the median pouch than is the right. The grouping of the tubules which arise from the lateral pouches is quite distinct except for the left medial and anterior left ‘ groups which have been rendered confluent by the vascular changes just discussed. The dorsal and ventral posterior clus- ters of the left side (fig. 45) are separate and the dorsal cluster which throughout early stages precedes the ventral one in develop- ment, is raised from the pouch surface and connected with it by a short broad pedicle. On the right side (fig. 46), the anterior right tubule group lies close to but is not fused with the right medial one. As on the opposite side, both dorsal and ventral clusters are distinct, and both are beginning to develop pedicles. These differences between the right and left parts of the pars hepatica mediana and between the lateral pouches were present to some small degree in the preceding stage, but are more notice- able in this specimen and become more marked during later development. They are due primarily to the unequal size of the omphalo-mesenteric veins. It is well known from the studies of Rabl (’92), Mayer (’89), Hammar (’93), and others, that in selachians there are at first two omphalo-mesenteric veins of almost equal size. Early in development however the right omphalo-mesenteric vein loses its connection with the area vasculosa and for a time ends blindly on the lateral wall of the yolk-stalk. Later a connection is formed between the posterior end of the right omphalo-mesenteric vein and the subintestinal vein by means of a channel lying to the right of the pancreas. During the period while the right omphalo- mesenteric vein ends blindly behind the blood from the yolk sac and from the subintestinal vein passes forward through the left omphalo-mesenteric vein alone, and this vessel in conse- quence becomes much enlarged. At this time the rotation of the gut from left to right about a longitudinal axis is in progress and the passageway for the vascular channel between the lateral hepatic pouch and the median hepatic pouch and fore gut is some- what larger on the left side than on the right. This condition is shown both in the anterior view of the model of an embryo DEVELOPMENT OF THE ELASMOBRANCH LIVER 365 10 mm. long and in figure 8 A, a cross section of the same specimen. The effect of the enlargement of the left omphalo-mesenteric vein upon the liver anlage has already been described in part and will be considered farther in describing later stages. Passing along the left side of the median hepatic pouch or the common bile duct it pushes this structure to the right and shifts the left V. subint. Fig.17 Frontal section of the liver and mid gut region of an Acanthias embryo 13 mm. long (H.E.C. 226). X 40. L.hep.p., lateral hepatic pouch; M.g., mid gut; M.hep.p., median hepatic pouch; S.v., sinus venosus; V.omph.l., left omphalo- mesenteric vein; V.omph.r., right omphalo-mesenteric vein; V.subint., subintes- tinal vein. hepatic pouch or left hepatic duct anteriorly and laterally. This process is shown in an early stage by figure 17. At the same time the vessel upon encountering the left segment of the anterior and expanded part of the median hepatic pouch breaks up into several trunks which pass below and above the obstruction. One of the larger trunks passes through the dorsal part of the vertical cleft between the right and left anterior tubule groups or hepatic THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO. 3 366 RICHARD E. SCAMMON rami and gradually enlarging this space presses the tubules apart until it forms one of the larger channels of the vein. The right omphalo-mesenteric vein, after establishing its pos- terior connection with the subintestinal vein, which it does when the embryo reaches a length of about 10 mm., grows rapidly, although it never equals in size that of the opposite side, and does HH Pdvet.med. yy Vomph.l. i); cyst. Tig. 18 Transverse section of an Acanthias embryo, 15.5 mm. long (S.C. 1). x 100. D.cyst., cystic duct. G.bl., gall bladder; P.duct.med., pars ductus medi- ana; R.hep.p., right hepatic pouch; S., sinusoids of right omphalo-mesenteric vein; St., stomach; V.omph.l., left omphalo-mesenteric vein. ; not affect the position of the biliary apparatus to any great degree. At first the right and left omphalo-mesenteric veins pass forward to meet in front of the anterior portion of the median hepatic pouch but in later stages the vessels become confluent behind and below the anterior part of the median hepatic pouch or its derivative, thus forming a large sinus which increases the effect already begun by the left vitelline vein, viz., shifting the common DEVELOPMENT OF THE ELASMOBRANCH LIVER 367 bile duct and cystic duct to the right and the left hepatic forward and laterally. This condition is shown by a frontal section of a much older embryo in figure 19. D.chol. Fig. 19 Frontal section of an Acanthias embryo 41 mm. long (H.E.C. 371). x 40. D.chol., ductus choledochus; D.cyst., cystic duct; D.hep.r., right hepatic duct; Int.y.s., internal yolk sac; Panc., pancreas; St., stomach; V.int., valvular intestine; V.omph.l., left omphalo-mesenteric vein. An embryo 20.5 mm. in length shows sufficient differentiation of the embryonic hepatic structures to permit the introduction of the adult terminology in describing them. As will be seen from a cross section through the anterior end of the gall bladder of a specimen of nearly the same stage (fig. 20), both lateral and me- » dian pouches are reduced in diameter and the tubule groups, in 368 RICHARD E. SCAMMON part at any rate, are connected with the latter by short ducts. A reconstruction of the biliary apparatus of this stage is shown in figures 47, 48 and 49. > duct.med. D.cyst. Fig. 20 Transverse section of an Acanthias embryo 19 mm. long (S.C. 2). X 100. D.cyst., cystic duct; G.bl., gall bladder; L.hep.p., lateral hepatie pouch; P.duct.med., pars ductus mediana. The pars ductus mediana, or as the structure may now be termed, the terminal part of the ductus choledochus, is tubular in form. The pars hepatica mediana is reduced in size antero- posteriorly and expanded laterally. The vertical cleft dividing it into right and left parts lies lateral to the left wall of pars duc- DEVELOPMENT OF THE ELASMOBRANCH LIVER 369 tus and is accentuated by the two short ducts which arise on either side of it. These ducts are the right and left medial hepatic rami and are the derivatives of the right medial and left medial tubule groups respectively. As yet they are very short and large ealibered and break up almost immediately into a number of hepatic tubules. Two tubules arising immediately ventral to the left medial ramus probably represent the remains of the ante- rior left tubule group whose fusion with the left medial group has been described. ‘Three additional tubules of large caliber arise from the anterior and ventral surface of the pars hepatica mediana. They may be derived from the right or left medial group but probably have arisen direct from the pouch wall after the main tubule groups were established. The left hepatic pouch or duct as we may now term the struc- ture, takes origin entirely from the lateral part of the posterior surface of the pars hepatica mediana, having been entirely sepa- rated from the pars ductus mesially. Thus it is already evident that the adult hepatic duct upon this side is made up of two ele- ments, a proximal and transverse part derived from the left part of the pars hepatica mediana and a distal and longitudinal part formed from the left lateral pouch proper. On the opposite side the pouch or duct arises, as in preceding stages, from the lateral surface of the pars ductus or common bile duct. A dis- tinct groove separates the anterior boundary of the right duct from the pars hepatica mediana. Also as in earlier stages the left duct is widely separated from the pars ductus while the right duct lies quite close to its opposite side. The condition of the — anterior left tubule group has already been described and on the right side the anterior group is connected with the main hepatic duct by a distinct neck, the anterior right hepatic ramus, which is directed dorsally. On the left side the dorsal posterior cluster or ramus is-‘no farther developed than before, but the ventral clustez possesses a short duct which extends posteriorly and bifurcates into upper and lower branches. On the right side the dorsal pos- terior cluster is represented by two ducts, the upper one being particularly prominent and directed posteriorly. The posterior ventral cluster arises from a very short broad diverticulum of the 370 RICHARD E. SCAMMON pouch and breaks up into five large tubules, the largest of which is directed posteriorly. In an embryo of 20.6 mm. (H.E.C. 1494, No. 28, Normal plate series) the hepatic ducts are so completely formed that their origin from pouches and tubule groups would hardly be surmised from the reconstruction of them seen in figure 50. The pars ductus mediana is not separable from the more posterior part of the ductus choledochus and the left part of the pars he- patica mediana and the left lateral pouch form together one duct, the left hepatic, which arises from the lateral surface of the ductus choledochus and curves first laterally and then backward. The distinct angle between the transverse and longitudinal parts of the duct is the point of union of the two elements which form it. The right medial hepatic ramus arises at the union of the left hepatic duct with the ductus choledochus. Immediately to the left of this is a smooth narrow segment of the left hepatic duct which lies between two branches of the left vitelline vein. ‘To the left of this segment lies a dorsal duct, the left medial ramus, and below and lateral to it is the anterior left ramus. These represent the anterior left and left medial tubule clusters respec- tively. The small tubules which are seen in the anterior view of the model arising between these two ducts are probably derived in part from both tubule groups which, it will be remembered, were somewhat fused in earlier stages. The right hepatic duct is much shorter than the left. It takes origin from the anterior end of the ductus choledochus and curves - backward at once. A very large dorsal ramus arises from its upper surface just distal to its connection with the ductus chole- dochus. ‘This is the anterior right hepatic ramus and corresponds to the tubule group of the same name in younger embryos. Aside from this and one small tubule arising from the anterior surface, the proximal part of the right hepatic duct is smooth. - Near its distal end it gives off several minor tubules. The dorsal ones represent the dorsal posterior tubule cluster while the ventral ones are derived from the ventral posterior tubule cluster as is also the most distal part of the duct itself. By this stage all the minor hepatic rami are established and no new tubules arise from DEVELOPMENT OF THE ELASMOBRANCH LIVER 371 the ducts direct, all further increase in hepatic parenchyma being due to growth in the hepatic trabeculae proper. The later histories of the major and minor hepatic ducts can be most easily undertaken separately. Up to the stage just de- scribed the gradual shifting of the anterior end of the ductus choledochus to the right has been a constant feature. This process becomes more noticeable with the distinct differentiation of the hepatic ducts and by the time the embryo reaches a length of from 25 to 28 mm., the anterior end of the duct may be so ro- tated that the original anterior surface faces the right and the left surface appears as the anterior one. The rotation though always present is not always so extreme as this type which is shown in figures 51 and 52. This process is brought about by a distinct enlargement of the sinus formed by the omphalo-mesen- teric veins which lies below and to the right of the ductus chole- dochus. ‘The rapid growth of this sinus is in turn due to changes in the hepatic trabeculae. Until the embryo reaches the length of from 25 to 28 mm. the trabeculae form a wide meshed network and the blood is able to flow around them through sinusoids of large caliber. From this stage on however the trabeculae in- crease rapidly in size and the surrounding sinusoids are reduced to extremely small vascular channels.’ The blood is thus directed in a large part into the omphalo-mesenteric veins and their caliber is considerably increased. The main hepatic ducts are markedly jaduenced by this rota- tion. The left one is carried forward until almost its entire length lies in the transverse plane of the embryo and only the dis- tal end with the tubules arising from it project backward while the right hepatic duct extends directly backward with little or no lateral course (figs. 21, 51, 52 and 54). Soon after the rotation of the anterior end of the ductus choledochus takes place the cystic duct, which until this time has joined with the ductus chole- dochus from below, is carried forward and upward along with the gall bladder until it joins with the anterior surface of the ductus choledochus and appears at first sight as an anterior extension of 5 This fact has already been recognized and recorded by Minot (’00). 372 RICHARD E. SCAMMON that structure. This process, illustrated in figures 22, 23, 24, 25 and 54, is brought about in part at least by the growth of the internal yolk sae and is discussed in more detail in the section upon the development of the gall bladder. A distinct forward arch appears in the proximal portion of the main hepatic ducts so that instead of entering the ductus choledochus at right angles to its longitudinal axis as in younger stage, they extend backward on either side of it for a short distance and then join with it very obliquely. This arch may be partially brought about by the hepatic .ducts being forced forward by the same agency as that influencing the cystic apparatus at this time but probably it is also due in part to the actual shifting backward of the ostia of the hepatic ducts along the ductus choledochus. Up to the stage represented by the embryo of 20.6 mm., the hepatic ducts were described with little difficulty for they pursue much the same course in all the specimens which were examined. However, the reduction in size of the distal end of the ductus choledochus and the shiftings which it and the hepatic ducts undergo modify considerably the position of some of the minor ducts and to varying degrees in different specimens. This applies particularly to the anterior left and right lateral rami and the left and right medial rami. The posterior dorsal rami remain fairly constant in position at the juncture of the transverse and posterior parts of their respective trunks, and the posterior ven- tral rami, while giving rise to new short minor sprouts in their course, seem fairly regular. Of the several types observed in embryos and specimens of the pup stage, the commonest and simplest one is that in which rotation of the ductus choledochus is present but not extreme and in which the embryonic arrangement of the rami is to a large extent retained. This form is illustrated by the graphic reconstruction of two embryos, one 28 mm. and one 41 mm. in length illustrated in figures 21 and 22. The variants from this type so far as observed have been such as might be expected from farther rotation of the anterior part of the ductus choledochus. If this process is extreme all those rami which arise from the anterior surface of the pars hepatica of an embryo 28 mm. long (H.E.C. 221). Xx 20. For abbreviations, see figure 22. Fig. 22 Graphic reconstruction (dorsal view) of the gall bladder and liver ducts of an embryo 41 mm. long (H.E.C. 371). 20. D.chol., ductus choledochus; D.cyst., cystic duct; D.hep.l., left hepatic duct; D.hep.r., right hepatic duct; G.Ob1., gall bladder; R.a.l., anterior left hepatic ramus; R.a.r., anterior right hepatic ramus; #.l.m., left medial hepatic ramus; R.r.m., right medial hepatic ramus; R.p.l., posterior left hepatic ramus; /.p.r., posterior right hepatic ramus. _ Fig. 23 Graphic reconstruction (dorsal view) of the gall bladder and liver ducts of an embryo 18 em. in length. X 10. For abbreviations, see figure 22. Fig. 24 Graphic reconstruction (dorsal view) of the gall bladder and liver ducts of an embryo 60 mm. in length (H.E.C. 409). X 20. For abbreviations, see figure 22. Fig. 25 Graphic reconstruction (dorsal view) of the gall bladder and liver ducts of an embryo 86 mm. in length (H.E.C. 410). X 20. Only the proximal part of the hepatic ducts are represented. For abbreviations, see figure 22. 373 374 RICHARD E. SCAMMON mediana and apparently even the anterior left ramus may be carried dextrally until they face the right. Apparently also any intermediate step between this extreme and the embryonic type described above may exist. When rotation first takes place and before the cystic duct begins to shift upward and forward the ostia of the minor anterior ducts remain quite close to that of the main left hepatic duct. With this change however the curve formed by the extreme anterior part of the ductus choledo- chus and the cystic duct is gradually obliterated so that the two ducts come to form together a slender tube which extends antero- posteriorly in frontal plane, and the ostia of the rami which for- merly were on the anterior surface of this curve come to lie on one side or the other of duct and are separated by its dorsal surface. Figures 51 and 52 are of a reconstruction of the biliary apparatus at a stage immediately after a distinct dextral rotation of the duc- tus choledochus has taken place and before the dorso-anterior migration of the cystic duct and gall bladder is very noticeable. Had this specimen continued its growth, if one may judge from reconstructions of later embryos, the cystic duct would probably be carried forward and upwards in such a way that it would inter- vene between the left medial ramus and the left main hepatic duct in such a way that it would receive the opening of the former on the right hand side and the latter on the left. Figures 53 and 54 of an embryo 33.1 mm. in length shows a somewhat later stage in which the cystic duct is being forced forward and the right medial ramus which formerly lay almost in the median line is being carried .over to the right side of the duct. Some reconstructions which illustrate the results brought about by the above processes may be illustrated here as they also show the changes which take place in the major ducts and gall bladder in the later periods of embryonic development. Figure 23 of a very late embryo 18 cm. in length sober a vari- ant in which the left medial ramus has remained attached to the main left hepatic duct but in which the right medial ramus arises from the ductus choledochus. Except for this change, the rami have remained in their early embryonic position. Figure ee DEVELOPMENT OF THE ELASMOBRANCH LIVER 375 24 of an embryo 60 mm. in length shows a specimen in which the rotation of the ductus choledochus to the right must have been very great indeed for it still shows an almost right angled curve in that direction at this late stage and the right medial, the left medial, and the anterior left rami all arise from it between the ostia of the cystic and left hepatic and the right hepatic duct. The figure of an embryo of 86 mm. (fig. 25) shows the most extreme case of the series in which both the left and the right anterior rami arise from the right hepatic duct, the latter almost at its ostium. I can only explain this case on the supposition that the posteriorly directed proximal segment of each the right and left main hepatic ducts is derived in part by a splitting off from the ductus choledochus and that the left medial ramus originally connected with an area of the right side of the ductus choledochus which was later incorporated in the distal end of the right lateral hepatic duct. That other variations in the arrangement of these hepatic rami may occur both in embryo and adult is very probable, but it is to be expected that they will all be of the same general type as those described above, viz., such as may be’ brought about by the progression of their ostia from left to right. Besides the variation in position of ostia, there are also noticeable ones in the size of the various elements. This seems to be compensatory in that the re- duction in extent of any element is accompanied by an increase in size and complexity of its neighbors. Occasionally one element may be replaced by two or more smaller ones and minor rami are commonly found irregularly-placed in regard to the larger ones. These minor sprouts as already stated arise either from tubules intermediate in position to the main tubule groups or from such tubules as arise after these groups are somewhat separated from the main ducts by pedicles. A scheme of the origin of the minor ducts or rami will be found in the general summary in table 3. 376 RICHARD E. SCAMMON III. DEVELOPMENT OF THE GALL BLADDER AND CYSTIC DUCT The gall bladder has been described in the first section of this paper as arising as a median outpouching of the anterior wall of the yolk-stalk immediately below the point where that struc- ture becomes continuous with the floor of the fore gut. The ven- tral zone of the lateral walls and the floor of this part of the gut at this early stage are a part of the hepatic area and that part of the anterior wall of the yolk-stalk from which the gall bladder arises is later incorporated in the gut behind the liver as the yolk- stalk becomes constricted antero-posteriorly. The time of appear- ance of the anlage of the gall bladder is somewhat later than that of the liver as has been noted by Laguesse (93) and Debeyra (09). It seems possible, from these observations, to regard the gall bladder in this form as derived from a secondary pouch which is formed in the floor of the archenteron immediately behind the liver pouch proper. If this interpretation be correct, an ante- rior shifting of the gall bladder by which its opening reaches its later position at the anterior end of the ductus choledochus in front of the openings of the hepatic ducts is to be expected. Evi- dences of such a shifting are not wanting. The early demarkation of the gall bladder has already been described in the preceding section, and has been seen to consist of the formation of dorsal longitudinal grooves and a posterior and ventral vertical one by which the gall bladder is cut off from the anterior wall of the yolk-stalk behind and remains attached to the liver pouch above by a transversely constricted neck which extends along its entire dorsal surface. The anterior end of this neck is less constricted and represents the cystic duct. These changes are illustrated in figures 33, 35 and 37, and are also shown in B of figure 26 below. These changes are even more marked in an embryo of 10 mm. illustrated in figures 39 and 40, and in C of figure 26. Along with them is a considerable lateral expansion of the gall bladder and a curious growth of its anterior wall which produces a pointed process which projects forward below the ventral wall of the me- dian hepatic pouch. The gall bladder in this and the preceding stages which follow its separation from the anterior wall of the DEVELOPMENT OF THE ELASMOBRANCH LIVER 37 ~I yolk-stalk behind lis a little to the left of the median line. This asymmetric position has already been discussed in Part I. It is associated with the rotation of the gut in connection with the formation of the spiral valve. When the liver reaches the stage represented in figures 41 and 42, of an embryo of 15 mm., the gall bladder is so far sepa- rated from it as to form a thick walled ovoid sac, the posterior half of which is rounded and free and the anterior end drawn out into a cone shaped projection. It is still broadly attached Fig. 26 A series of semi-schematic figures of the development of the gall bladder and cystic duct. A, embryo of 7mm.; 8B, embryo of 7.5mm.;C, embryo of 10mm.; D, Embryo of 15.5 mm.; #, embryo of 19mm.; F, embryo of 20.6 mm.; G, embryo of 33.1 mm. All from plastic reconstructions with the exception of A, which is based upon longi-sections. D.chol., ductus choledochus; D.cyst., cystic duct; D.hep., hepatic duct; G.bl., gall bladder; Hep.d., hepatie diverticulum. to the median liver pouch above. The posterior end extends downward so that the long axis of the sac is no longer parallel with that of the median liver pouch above it. The gall bladder in an embryo of 15.5 mm. (figs. 45 and 46 and D, fig. 26) is smaller relatively, and the connection between it and the liver above is drawn out into a short broad stalk, the cystic duct. The pointed anterior extremity is less noticeable than in the two preceding specimens and from this time on no evidences are seen of it. In this specimen, also, the gall bladder 378 RICHARD E. SCAMMON lies to the left of the median line and the cystic duct is inclined to the right as well as upward to meet the median hepatic pouch or ductus choledochus. At 19 mm., the gall bladder is widely separated from the ductus choledochus and inclined at a distinct angle to it. The cystic duct is decidedly elongated and extends upward to the right and decidedly forward to meet the ductus choledochus. This elonga- tion is due in part to the increase in size of the left omphalo- mesenteric vein, which lies in this region between the gall bladder below and the ductus choledochus above, and perhaps also in part to the great growth of the hepatic trabeculae which takes place at this time. Probably the increased length of the duct is derived in part at least from the lower part of the ductus chole- dochus from which the anterior end of the cystic duct seems to be separating. This separation is peculiar in that it takes place mesially more rapidly than laterally, thus forming a distinct pocket which is bounded below by the cystic duct, above by the floor of the ductus choledochus and laterally by the sides of these structures which are still confluent. This formation is only temporary.. . . At 20.6 mm. (fig. 50, and fig. 26, F) the cystic duct is still more elongated and joins the floor of the ductus choledochus nearly at right angles to it. The gall bladder is shifted downward until its long axis is almost in the vertical plane of the embryo and at right angles to the ductus choledochus. It is drawn out until the sae appears as little more than the expanded end of the cystic duct. The shifting of the ductus choledochus and the rotation of its anterior end, described on page 371, now takes place, and the cystic duct and a part of the gall bladder share in this displace- ment. The posterior half of the gall bladder remains almost in the position occupied in the preceding stage or is shifted a ~ little dorsally, while the anterior half and the cystic duct form together an abruptly curved tube which extends to the right and dorsally. In this way there is formed a distinct flexure in the middle part of the gall bladder. This is illustrated by an embryo * 28 mm. in length (figs. 51 and 52). DEVELOPMENT OF THE ELASMOBRANCH LIVER 379 V.omph.t. Valvular intestine. Fig. 27 Frontal section of an Acanthias embryo 86 mm. long (H.E.C. 410). x 40. D.chol., ductus choledochus; G.bl., gall bladder; Panc., pancreas; St., stomach; V.omph.l., left omphalo-mesenteric vein. Along with the changes described above comes a very extensive alteration in both the form and position of the cystic apparatus which is apparently produced by the development of the internal yolk sac. This structure begins to develop from the vitelline duct just internal to the ventral body wall in embryos from 25 to 30 mm. in length. An early stage is seen in section in figure 17. It grows rapidly and its anterior end pushes forward be- tween and below the stomach and left and cystic lobes of the liver on one side and the right lobe of the liver on the other. The frontal section shown in figure 27 illustrates its size and relations in an embryo 60 mm. in length. 380 RICHARD E. SCAMMON In this way the yolk sac presses upon the right side and lower surface of the gall bladder. The first effect of this pressure is to force the posterior part of the gall bladder upward so that this structure retraces in part the path of downward extension which it followed in earlier stages (figs. 22, 23, 24, 25 and 26 G). At the same time the gall bladder is elongated and somewhat flat- tened vertically and its anterior end is pushed upward and for- ward. At first this only causes an abrupt curve in the cystic duct (fig. 54), but as this process is continued the gall bladder is finally forced anteriorly and dorsally beyond the distal end of the ductus choledochus and the cystic duct proceeds backwards over its dorsal surface to join that structure. In this way the ostium of the cystic duct which was originally in the floor of the ductus choledochus is rotated to its anterior surface and the two ducts together form one continuous tube sometimes called the ductus cystocholedochus, the juncture of the two elements of which is at the ostia of the hepatic ducts. This is also illustrated in figures 22, 23, 24, 25 and 26 G. ’ IV. DEVELOPMENT OF THE DUCTUS CHOLEDOCHUS The development of the ductus choledochus is better known than that of any other part of the biliary apparatus in elasmo- branchs. For our information in regard to the earlier stages we mainly have to thank Hammar (’93), Brachet (’96), Mayr (97) and Choronschitzky (’00) while the iater stages have been most successfully studied in connection with the development of the spiral valve by means of the reconstruction method by Rickert (’96, ’97). The development of this structure in Acanthias has been to some extent described incidentally in connection with the account of the early stages of the liver and of the hepatic and cystic ducts and gall bladder in this paper so that only a short summary need be given here. In Acanthias, the ductus choledochus when fully formed is a complex consisting of three embryonic elements: (1) a proximal or posterior segment derived from the floor of the duodenum and DEVELOPMENT OF THE ELASMOBRANCH LIVER 381 valvular intestine; (2) a middle segment formed from the ‘pars ductus lateralis’ of the primitive lateral hepatic diverticula; (3) a distal and anterior segment which is differentiated from the posterior portion or ‘pars ductus mediana’ of the secondary me- dian hepatic pouch. Thus the middle and distal portions of the duct are both derived from the primitive lateral diverticula, at a very early stage and are truly hepatic in origin while the proximal or posterior part is formed from the archenteron at a much later stage after the duodenum and valvular intestine. In the fully formed fish the latter segment forms by far the greater part of the duct being represented by practically all the extra hepatic portion. The differentiation of the posterior part of the lateral hepatic diverticula into the pars ductus lateralis has already been de- scribed in Part I, as well as the early demarkation of the pars ductus mediana from the median hepatic pouch. At 10 mm. the pars ductus lateralis forms the slightly expanded ventral half of the rather constricted segment of archenteron which connects the median hepatic pouch and the gut above it with the yolk- stalk and mid gut posteriorly (figs. 39 and 40). The pars ductus lateralis is thus continuous anteriorly and below with the gall bladder and median pouch and above with the dorsal part of this stalk which later forms the duodenum. On the dorsal surface of the median pouch (fig. 28) is an oblique ridge which extends from its left anterior to right posterior angle and maps off the area of this structure which later becomes the distal part of the ductus choledochus. At 15 mm. (figs. 41 and 42) both the median hepatic pouch and the pars ductus lateralis behind it are completely cut off from the gut above. The latter now forms a short wide duct about 0.1 mm. in length which extends back- ward a little obliquely from left to right and joins with the floor of the gut a little to the right of the median line. In cross section the duct is equal in diameter to that segment of gut, the duode- num, with which it joins and is triangular in outline with the apex of the triangle directed upward. It thus shows a trace of the pouch-like structure from which it arises but at a little later stage it becomes circular or oval in cross section. The pars duc- THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO. 3 382 RICHARD E. SCAMMON tus mediana is still sac-like and like the duct behind it is inclined a little from left to right. In 15.5 to 18 mm. embryos the part of the duct derived from the pars lateralis is nearly twice as long as in the above specimen (figs. 45 and 46) and extends directly backward joining the pos- terior end of the duodenum in the median line. Its diameter is about 0.1 mm., being distinctly less than that of the earlier stage and about one-half that of the gut which it joins. The pars duc- tus mediana is rapidly taking on a duct-like form although this feature is somewhat obscured by the large ducts which arise from ive Fig. 28 Dorsal view of a reconstruction of an Acanthias embryo 10 mm. long (S.C. 20).. X 36. Lateral and anterior views of this model are shown in figures 38 and 39. A, anterior part; B, posterior part of ridge marking the formation of the anterior part of ductus choledochus from the pars ductus medialis of the median liver pouch. F.g., fore gut; L.hep.p., lateral hepatic pouches which are cut away dorsally; Sp.v., spiral valve; Y.s., yolk stalk. Fig. 29 Reconstruction of the mid gut region of an Acanthias embryo 20.5 mm. long (8.C. 5). x 50. Duo., duodenum; D.chol., ductus choledochus; D.panc., pancreatic duct; D.vit., vitelline duct; V.int., valvular intestine. In the stages which now follow, as is shown in figures 51, 52 and 54, the duct rapidly increases in length due to the elongation of its extra-hepatic portion. This growth seems to be derived from the floor of the digestive tube for there extends in the floor of the gut posterior to the ostium of the duct a deep groove which is lined with epithelium of the same nature as that of the duct itself. At 15.5 mm., the duct joins the gut near the posterior end of the duodenum. At 20.5 mm., as is shown in figure 29, this opening lies at the point where the duodenum becomes continuous DEVELOPMENT OF THE ELASMOBRANCH LIVER 383 with the valvular intestine in the angle between the latter struc- ture and the vitelline duct. The duct lies below and to the right of the median line of the duodenum, but as this structure is con- tinuous only with the left side of the valvular intestine (the right side of which is continuous with the vitelline duct) the ductus choledochus joins the valvular intestine directly in the median line of the gut. From this time to until the embryo reaches a length of approxi- mately 25 mm. the duct grows steadily backward and its ostium remains in the median line. It grows past the opening of the vitelline duct passing it to the left but never overtakes the ostium of the pancreatic duct. When the embryo reaches the length of about 25 mm., however, the duct becomes involved in the twisting process of the spiral valve and is carried to the left. At 28 mm. its opening is in the middle of the right side of the valvular intestine and at 37 to 48 mm. it hes at the junction of the superior and right surfaces. In embryos 60 to 80 mm. long, the duct enters the intestine on its dorsal surface at the edge of the first turn of the spiral valve, and in the new-born fish the opening lies at the junction of the left side with the dorsal surface of the intestine. These changes in position as well as the comparative diameter of the duct at differ- ent stages are shown in table 2. Along with the elongation of the ductus. choledochus come several modifications of its position besides the posterior shifting just described. The intrahepatic portion is affected by the growth of the internal yolk sac and by the vascular changes already dis- cussed in connection with their effect upon the position of the hepatic and cystic ducts. This part of ductus choledochus is first arched upward in a stiff almost semi-circular curve by the formation of a venous sinus below it and its extreme anterior end is rotated to the right. These changes are shown in figures 51, 52 and 54. In later stages and in the new-born fish these changes are less noticeable, being probably compensated by the . growth of the hepatic parenchyma, but in the new-born fish as well as the adult the distal end of the duct lies distinctly to right of the median line. As the duodenal flexure is developed, the 384 RICHARD E. SCAMMON middle part of the duct is also directed sharply ventrally along with the change in position of the segment of the gut which it joins. This ventral curve appears in embryos of 18 to 20 mm. and is at first a gradual one but becomes sharper and more pro- Fig. 30 A series of transverse sections of Acanthias embryos of different ages at the level of the ostium of the ductus choledochus. A, embryo 15.5 mm. long (S.C. 1); B, embryo 20.6 mm. long (H.E.C. 1494); C, embryo 32.2 mm. long (H.E.C. 1662); D, embryo 180 mm. long (S.C. 51) all except D X 28; D X 8. D.chol., ductus choledochus; D.panc., pancreatic duct; D.vit., vitelline duct; Duo., duo- denum; Panc, pancreas; St., stomach. nounced in older embryos. In the course of this curve the duct is also pushed to the right by the vitelline vein which lies beside it. Minor short flexures appear in later stages near the anterior and . posterior ends of the duct and in its duodenal curve as well. They are not constant in position, shape or number. The duct DEVELOPMENT OF THE ELASMOBRANCH LIVER 385 is much reduced in diameter between the time when it is first formed and when it begins it growth backward along the base of the valvular intestine. After this phase begins there is but little change in caliber. In the intrahepatic and preduodenal regions the duct is circular in trans-section, posteriorly it is flattened transversely until elongately oval in trans-section. Table 2 shows numerically the growth of the duct and the chang- ing relations of its ostium. Some error is doubtless introduced in the calculation of the length of the duct from cross sections as is done here, but Acanthias embryos of 18 mm. and over shrink comparatively little in imbedding and that shrinkage seems to be symmetrical so that the value of the figures is not seriously im- paired. The ‘extra-hepatic length’ is taken from the point where the duct turns sharply downward immediately after leaving the liver to the point where its lumen becomes continuous with that of the gut. It is not possible to determine this point exactly in specimens under 20 mm. in length so only the total length of the duct has been given in such eases. TABLE 2 Growth and relations of the ductus choledochus RELATION|RELATION LENGTH OF DUCT OF OF DIAM- | OSTIUM | OSTIUM een ETER TO TO POSITION OF OSTIUM IN ARIS) SUSE SIRO IS CGE a OF OST:UM | OSTIUM INTESTINAL WALL BRYOS| Tntra | Extra Total | DUCT |OF VITEL-| OF PAN- hepatic| hepatic LINE CREATIC pUCcT DUCT mm. mm. mm. mm. mm. mm. mm, HEC. 227...) 15.0 0.11 0.15 0.23 ant.) 0.40 ant.| Entire ventral surface SUS ale see Lao 0.22 0.11 | 0.20 ant.| 0.32 ant.) Entire ventral surface Se Che aerate eee 19.0 | 0.16 0.9 0.14 ant.) 0.33 ant.) Entire ventral surface H.E.C. 1494. 20.6 0.22 0.18 0.40 0.8 Same | 0.12 ant.! Mid-line of ventral sur- plane. face SB: G5 1492) 247 || 0.10 0.32 0.42 0.8 |0.10 post.) 0.35 ant.| Junction right and mid- | dle thirds of ventral surface B..E.C. 1357. .} 28.0 0.70 0.62 0.72 0.5 |0.40 post.| 0.25 ant. Junction lower and mid- dle thirds of right sur- face 0.60 post.| 0.29 ant.| Junction lower and mid- dle thirds of right sur- face H.E.C. 363...| 37.0 0.36 1.08 1.44 0.7 |0.80 post.) 0.46 ant.) Junction right and dorsal surfaces. .76 ant.| Junetion right and dor- sal surfaces 2.4 post.| 1.2 ant.| Mid-line of dorsal surface ao H.E.C. 1652..)| 32.2 0.16 0.86 1.62 0. _ BiG) Tis). 47.3 | 0.72 | 1.10 | 1.82 | 0.7 [0.91 post. H.E.C. 1882..| 95.0 1.20 5.0 6.2 0. | ag 386 RICHARD E. SCAMMON V. GENERAL SUMMARY The observations here recorded may be summarized as follows: 1. The liver arises in Acanthias embryos of from 20 to 25 seg- ments as a pair of shallow lateral diverticula from the lateral walls of the ventral half of the gut. These diverticula extend both behind and in front of the anterior wall of the yolk-stalk. 2. The growth of the fore gut posteriorly through the coales- cence of the lateral walls of the yolk-stalk causes the lateral hepatic diverticula to lie mainly in front of the anterior wall of the yolk- stalk in later stages. 3. The median veritral liver pouch described by Balfour and others is, in Acanthias at least, a secondary structure produced by the fusion of the anterior ends of the primary lateral diverticula. 4. Three distinct secondary parts are derived from each primi- tive lateral hepatic diverticulum. The anterior portion goes to the median hepatic pouch. The upper part of the middle por- tion forms the lateral hepatic pouch. The posterior part goes to form a posterior connecting segment between the liver and gut. This has been termed the pars ductus lateralis and later becomes a part of the ductus choledochus. 5. At an early stage the liver shares in the left to right rotation which produces the spiral valve in the intestine and the lateral vitelline groove in the yolk stalk. 6. The median hepatic pouch is somewhat differentiated into two parts: an anterior one, the pars hepatica mediana to which the lateral pouches are mainly attached and which gives rise to hepatic trabeculae, and a posterior one, the pars ductus mediana, which forms the anterior part of the ductus choledochus. 7. The anterior part of the left hepatic duct is formed from the left hepatic pouch and the left part of the pars hepatica mediana. The anterior part of the right hepatic duct is formed from the right hepatic pouch alone. 8. The hepatic ducts and ductus choledochus are very markedly rotated to the right about a vertical axis in a comparatively late stage. This rotation is probably due to the great growth of the Jeft omphalo-mesenteric vein and the formation of a venous DEVELOPMENT OF THE ELASMOBRANCH LIVER 387 sinus below and to the left of the anterior end of the ductus choledochus. 9. The difference in size of the right and left omphalo-mesen- teric veins may be due to the longitudinal rotation of the gut mentioned in section 5 of this summary. By this rotation the space between the left hepatic pouch and the median pouch and gut is somewhat increased while the corresponding space on the opposite side is decreased. 10. A third shifting of the duct system and the gall bladder forward and upward occurs at a much later stage. It is probably brought about through the great increase in size of the internal yolk sac. 11. The minor hepatic ducts arise as the elongated pedicles of definitely placed groups of hepatic tubules. These tubule groups are differentiated at the time when the liver loses its dorsal con- nection with the fore gut. 12. The variations in position of the minor hepatic ducts in the adult depend upon the degree of shifting of these tubule groups at the time when the main hepatic pouches are differentiated into hepatic ducts and when the rotations mentioned in sections 5 and 8 take place. 13. The gall bladder appears much later than do the primary lateral hepatic diverticula. It arises posterior to the hepatic anlage as a distinct evagination of the gut at the juncture of the floor of the fore gut and anterior wall of the yolk sac, and its inti- mate connection with the liver duct system is acquired secondarily. 14. The gall bladder loses its dorsal and posterior connection with the gut and is shifted forward and downward until its great- est axis is in the transverse plane of the body. In this way its connection with the ductus choledochus comes to lie in front of that of the lateral hepatic ducts. 15. Subsequently the gall bladder is again shifted upward and forward so that the cystic duct passes backward and downward to join the ductus choledochus. This is brought about by the agency mentioned in section 10. 16. The ductus choledochus is formed from three distinct elements: the anterior part from the pars ductus mediana, a deriva- 388 RICHARD E. SCAMMON tive of the secondary median hepatic pouch; the middle part from the pars ductus lateralis, a derivative of the posterior part of the primary hepatic diverticula, and a posterior part formed from the’floor of the duodenum and valvular intestine. 17. Finally, the results of this study may be summarized in tabular form as given below, starting with the sources of the he- patic apparatus on the left hand side, and ending with end results of their evolution on the right. In this table the minor ducts are arranged in the way that they seem to occur most frequently in the late embryo or new-born fish. To this table should be added the statement that all those embryonic structures which are in- cluded under the term ‘pars hepatica’ give rise to hepatic trabe- culae as well as to the conducting structures which are listed here. 389 DEVELOPMENT OF THE ELASMOBRANCH LIVER JOPPPTF TED | jonp asda | a} Toh sTy s\asina strolhe BORO D OCI DELI eC PE OOO ONO CO COON UO DOOD CBO DOGooa gag O00 C UOTPVUISVAD atysKo SMUG USI Oleg Uy te on JYSII LOLoUYy snued 1o11e4sod [es1oq °° * *1eysnyo [es10g eanona yonod LUWE DO 10 GC ou LOUETT |p sien neeneet nom cents Vy sta o1yedey Setar Patanastee TOPSN fo Sn gonp qaed [eqsiq IO1I94SOg VS (SI[B.107B] [eryue A : o1yedoy voryedoy qYSIY se ew wee “qaied [BUIIXO1g COCO eOudett OnciceOeoecn oer cl nO tlosmermd Oho OO Oo Apog S18q) SONDIN EAT GIRS YE QKONG RS MD yea Oe Ma SRO SCH ey) soyonod IOTIO}UV oredey SNUIBI LoI10ySsod [es1oq**" * Taqsnyo eaaaae yonod [8107 e'T ° Ie EtO Ci Garatrecronrs aT oneday ayo so) es ret oedey tourpy | ** *1038n7o eee man ; hee oyedoy a = hae 7e Say [e139 A need | [e104e'T] 4 Pp viv viele q1ed 0} BIPOULI0} UT BFipiye’ fais acial vi costae) in} (ellegiey a Meee (ey tefr a ixelrch eke emeN che M cata me Mee fie Apog J j SBC OU WOT | aaa tere Sage shat? Arle qivd [BUIIXOIg’***° ES apis yay ) snyoop 2 -afoyo snjyonp oy} Jo pus [eqstp 944 Se ADO, ee I TOUT a2 eccitsmedomcien = meme apis WY S1Yy Boryedoy jo javd ]]BUIs B SUTUIIOJ Sv pailepIs ; : aes ' area yonod -uoo9 oq Avul ‘podojoaep ATVYSISs yng dee > UBIPOUL SNUIBI O1Jedoy [VIpeul JjoT [eIpew doy sdnois | asi IOI4yUYy SUED Oedoy (eIpoUL ysis 0 ee es [BIpew YSIYy aynqny, J SNYPOPOND | ao: nero csains cole indie) een oe a a eR Te snyong yivd 0} ¥Ipoutie}Uy SI[BIOJV] SHJONp sieg Oo Gta es “qaed [euIxolg oh ie) (er wii is: [ei e.(e (8) 6 \e se, 6) ea) |e) .¢)0)\e (0) 0) «)(sihe)"e\16) 16,0) 161.0 (@)\ulisvele) aay (eke) (616) 1011¢)'s)'eije)) 6! 08) 676/16) 8) a: eee ew eee oUuT}Se} -UI Iv[NA[VA Jo Javd AOIIe}UB PUe WNUepoNp jo OOH WoOdJ poAltop yonq’**”° “7 -gn3 pry _— — —_ RA, Pn aa a soinjonays SoInjons4s D1UOAIQUIO OY vIPSUIIE}UT soimjoniys pedojeasp AN y oto AIquie SATOMI SDIYJUDIP Ur sainjonijs oyoday fo urihrso ay) fo hanuungy ¢ GTaVL 390 RICHARD E. SCAMMON BIBLIOGRAPHY Batrour, F. M. 1876 The development of elasmobranch fishes. From B. to G. Journ. Anat. and Phys., vol. 10. Bracuet, A. 1896 Recherches sur le développement du pancreas et du foie. (Selaciens, Reptiles, Mammiferes). Journ. d. l’Anat., tom. 32. 1897 Die Entwickelung und Histogenese der Leber und des Pancreas. Ergebnisse d. Anat. u. Entwickelungsges. Bd. 6. Braus, H. E. 1896 Untersuchungen zur vergleichenden Histologie der Leber der Wirbeltiere. Semon. Zool. Forschung in Australien, 4 Lief. CuHoronscHitzky, B. 1900 Die Entstehung der Muiltz, Leber, Gallenblasse, Bauchspecheldruse und des Pfortadersystems bei den verscheidenen Abteilungen der Wirbeltiere. Anat. Hefte, Bd. 13. Deseyra, A. 1909 Le foie, est-il d’origine Endodermique ou mésodermique. Biob. anat., tom. 19. Goprert, E. 1893 Die Entwickelung des Pankreas der Teleostier. Morph. Jahrb., Bd. 20. Hammar, J. A. 1893 EHinige Plattenmodelle zur Beleuchtung der friiheren Em- bryonalen Leberentwickelung. Nova Acta Reg. Soc. Sci. Upsala, Ser. 3, also Arch. Anat. Entwicklungsges. 1897 Ueber einige Hauptziige der ersten embryonalen Leberentwickel- ung. Anat. Anz., Bd. 13. Hou, J. F. 1897 Ueber den feineren Bau der Leber bei den niederan Wirbel- tieren. Zool. Jahrb., Anat., Abt., Bd. 10. LacuesseE, E. 1894 Développement du pancréas chez les Sélaciens et chez les vertébrés en general. Biob. anat., tom. 2. Leypic, F. 1852 Beitrage zur mikroskopischen Anatomie und Entwicklungs- geschichte der Rochen und Haie. Leipzig. Mayer, P. 1889 Ueber die Entwicklung des Herzens und der grossen Gefiss- stamme bei den Selachiern. Mitt. zool. Stat. Neapel., Bd. 7. Mayr, J. 1897 Ueber die Entwickelung des Pancreas bei Selachiern. Anat. Hefte, Bd. 8. Minot, ©.S. 1900 Ona hitherto unrecognized form of blood circulation without capillaries in the organs of Vertebrata. Pro. Boston Soc. Nat. Hist., vol. 29. Preer, H. 1902 Die Entwicklung von Leber, Pankreas und Milz. Historisch- j kritische Studie. Freiburg. (Diss.) Ratuke, H., 1827 Beitrige zur Geschichte der Thierwelt. Vierte Abtheilung. Danzig. Rasx, C. 1889, 1892, 1896 Theorie des mesoderms. Morph. Jahrb., Bds. 15, 19, 24. Also: Printed separately, Leipzig. 1892 Ueber die Entwicklung des Venensystems der Selachier. Festschr. zum 70. Geburtstag R. v. Leuckarts. Rickert, J. 1896 Spiraldarmentwickelung von Pristiurus. Verh. d. X. Ver- sammlung d. Anat. Ges. zu Berlin. : 1897 Ueber die Entwickelung des Spiraldarms bei Selachiern. Arch. Entwickelungsmechanik, Bd. 4. Scammon, R. E. 1911 Normal plates of the development of Squalus acanthias. Hefte 12, Normentaf. d. Ent. d. Wirbeltiere. Jena. Weser, J. A. 1903 L’origine des glandes annexes de |’intestine moyen chez le vertébrés. Arch. d’Anat. Mier., tom. 10. PLATES [i> Oa, a hs te | en, ) en ih oy ra he PLATE 1 EXPLANATION OF FIGURES 31 Lateral view of a reconstruction of a part of the archenteron of an Acanthias embryo 6.4 mm. long (S.C. 19). X 100. 32 Anterior view of the reconstruction seen in figure 31. > 100. 33 Lateral view of a reconstruction of the hepatic region of an Acanthias embryo 7.5 mm. long (H.E.C. 1503). X 75. 34 Anterior view of the reconstruction seen in figure 33. 35 Ventral view of a reconstruction of the hepatic region of an Acanthias embryo 9 mm. long (H.E.C. 1495). X 75. 36 Anterior view of the reconstruction seen in figure 35. X 75. 37 Ventral view of a reconstruction of the hepatic region of an Acanthias embryo 7.5 mm. long (S.C. 15). X 75. 38 Anterior view of the reconstruction seen in figure 37. X 75. B.en., blastodermic entoderm L.hep.p., lateral hepatic pouch D.cyst., eystie duct M.hep.p., median hepatic pouch F.g., fore gut T.a., anlagen of hepatic tubules G.bl., gall bladder Y.s., anterior wall of yolk stalk Hep.d., hepatic diverticulum 392 DEVELOPMENT OF THE ELASMOBRANCH-LIVER PLATE 1 RICHARD E. SCAMMON Hep.d. B. en. 32 393 PLATE 2 EXPLANATION OF FIGURES 39 Anterior view of a reconstruction of the liver of an Acanthias embryo 10 mm. long (S.C. 20). > 100. 40 Left lateral view of the same reconstruction. > 100. F.g., fore gut | P.hep.m., pars hepatica medialis G.bl., gall bladder T.a., anlagen of hepatic tubules L.hep.p., \ateral hepatic pouch Y.s., yolk stalk M.hep.p., median hepatic pouch 394 DEVELOPMENT OF THE ELASMOBRANCH LIVER, PLATE 2 RICHARD E. SCAMMON 395 THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO. 3 PLATE 3 EXPLANATION OF FIGURES 41 Right lateral view of a reconstruction of the liver of an Acanthias embryo 15 mm. long (H.-C. 227). x 150: 42 Left lateral view of the same reconstruction. D.chol., ductus choledochus T.d.l., dorsal cluster, posterior left G.bl., gall bladder tubule group L.hep.p., left hepatic pouch T.v.l., ventral cluster, posterior left R.hep.p., right hepatic pouch tubule group St., stomach T.r.m., right medial tubule group T.a.l., anterior left tubule group T.d.r., dorsal cluster, posterior right T.a.r., anterior right tubule group tubule group T.l.m., left medial tubule group T.v.r., ventral cluster, posterior right tubule group 396 DEVELOPMENT OF THE ELASMOBRANCH LIVER PLATE 3 RICHARD EB. SCAMMON 397 PLATE 4 EXPLANATION OF FIGURES 43 Anterior view of a reconstruction of the liver of an Acanthias embryo 15.5 raatials Woyayes ((SGq Wy >< 10) 44 Anterior view of the reconstruction illustrated in figures 41-42. G.bl., gall bladder St., stomach T.a.l., T.l.a., anterior left tubule group T.a.r., anterior right tubule group T.d.l., dorsal cluster, posterior left tubule group T.d.r., dorsal cluster, posterior right tubule group T.l.m., left medial tubule group x 150. T.p.l., posterior left tubule group— the separate clusters cannot be seen in this view of figure 48. T.r.m., right medial tubule group T.v.l., ventral cluster, posterior left tubule group T.v.r., ventral cluster, posterior right tubule group DEVELOPMENT OF THE ELASMOBRANCH LIVER PLATE 4 RICHARD E. SCAMMON 399 PLATE 5 EXPLANATION OF FIGURES 45 Left lateral view of a reconstruction of the liver of an Acanthias embryo 15.5 mm. long (S.C. 1). X 100. 46 Right lateral view of a reconstruction of the same reconstruction. D.chol., ductus choledochus D.cyst., cystic duct Duo., duodenum G.bl., gall bladder St., stomach T.a.l., anterior left tubule group T.a.r., anterior right tubule group T.d.l., dorsal cluster, posterior left tubule group x 100. T.d.r., dorsal cluster, posterior right tubule group T.l.m., left medial tubule group T.r.m., right medial tubule group T.v.l., ventral cluster, posterior left tubule group T.v.r., ventral cluster, posterior right tubule group 400 DEVELOPMENT OF THE ELASMOBRANCH LIVER PLATE 5 RICHARD E. SCAMMON Tal. 401 PLATE 6 EXPLANATION OF FIGURES 47 Left lateral view of a reconstruction of the liver of an Acanthias embryo 20.5 mm. long (S.C. 5). 75. 48 Right lateral view of the same object. X 75. D.chol., ductus choledochus T.d.r., dorsal cluster, posterior right D.cyst., eystie duet tubule group G.bl., gall bladder T.v.l., ventral cluster, posterior left L.hep.d., left hepatie duct tubule group T.a.l., anterior left tubule group » T.v.r., ventral cluster, posterior right T.a.r., anterior right tubule group tubule group T.d.l., dorsal cluster, posterior left tubule group 402 DEVELOPMENT OF THE ELASMOBRANCH LIVER PLATE 6 RICHARD E. SCAMMON otale 403 PLADE o7 EXPLANATION OF FIGURES 49 Anterior view of the reconstruction shown in figures 47 and 48. iD: 50 Anterior view of a reconstruction of the gall bladder and liver ducts of an Acanthias embryo 20.6 mm. long (H.E.C. 1494). D.chol., ductus choledochus D.cyst., cystic duct D.hep.l., left hepatic duct D.hep.r., right hepatic duct G.bl., gall bladder T.a.l., anterior left hepatic ramus or tubule group T.a.r., anterior right ramus or tubule group T.p.l., dorsal cluster, posterior left tubule group, which later forms the posterior left ramus 404 x 75. T.d.r., dorsal cluster, posterior right tubule group T.l.m., left medial ramus or tubule group T.r.m., right medial ramus or tubule group T.v.l., minor tubules of ventral left tubule group T.v.r., minor tubules of ventral right tubule group DEVELOPMENT OF THE ELASMOBRANCH LIVER RICHARD E. SCAMMON [der D.chol. 405 PLATE 7 PLATE 8 EXPLANATION OF FIGURES 51 Left lateral view of a reconstruction of a part of the gut and the liver ducts and gall bladder of an Acanthias embryo 28 mm. long (S.C. 6). X 50. 52 Antero-ventral view of the same reconstruction. 40. D.chol., ductus choledochus Panc., pancreas D.cyst., cystic duct. R.a.l., anterior left hepatic ramus D.hep.l., left hepatic duct R.a.r., anterior right ramus D.hep.r., right hepatic duct R.l.m., left medial ramus D.panc., pancreatic duct R.p.l., posterior left ramus D.vit., vitelline duct R.r.m., right medial ramus Duo., duodenum St., stomach G.bl., gall bladder V.int., valvular intestine 406 i PLATE > \ THE ELASMOBRANCH LIVEI RICHARD E. SCAMMON OF DEVELOPMENT }oy2"q IAG PLATE 9 EXPLANATION OF FIGURES 53 Anterior view of a reconstruction of the gall bladder and liver ducts of an Acanthias embryo 33.1 mm. long (S.C. 8). 50. 54 Ventral view of a reconstruction of the gall bladder, liver ducts, and a part of the gut of the same embryo. 50. D.chol., duetus choledochus R.a.r., anterior right ramus D.cyst., cystic duct R.d.l., dorsal posterior left ramus D.hep.l., left hepatic duct R.l.m., left medial ramus D.hep.r., right hepatie duct R.p.l., posterior left ramus D.vit., vitelline duct R.r.m., right medial ramus Duo., duodenum St., stomach G.bl., gall bladder V.int., valvular intestine R.a.l.,R.a.m.,anterior left hepatic ramus 408 DEVELOPMENT OF THE ELASMOBRANCH LIVER PLATE 9 RICHARD E. SCAMMON 409 THE FASCICULUS CEREBRO-SPINALIS IN THE ALBINO RAT S. WALTER RANSON The Anatomical Laboratory of the Northwestern University Medical School TEN FIGURES It is well known that the fasciculus cerebro-spinalis, more commonly called the cortico-spinal or pyramidal tract, does not occupy the same position in the spinal cord in all orders of mammals. But, according to the animal which is being studied, it may be found in any one or two of the three funiculi of the cord. From its constant position in the ventro-medial portion of the medulla it passes in the rat to the opposite posterior funicu- lus of the spinal cord; while in the mole it runs without decussa- tion into the anterior funiculus of the same side. In the cat it decussates into the opposite lateral funiculus, while in man a part of the fibers go over into the opposite lateral funiculus and a smaller part run without decussation into the homolateral ante- rior funiculus. . There have been published a large number of articles dealing with such variations in the position of the pyramidal tract, and with the corresponding variations in the pyramidal decussation; but very little attention has been paid to the character of the fibers of which this fasciculus is composed. A study of sections of the spinal cord of the rat, guinea-pig, rabbit and cat prepared by the puridine silver technique has brought to light great differences in the pyramidal fibers in these different animals. The varia- tions in the size of the axons and in the degree to which the myelin sheaths are developed are no less striking, and probably more significant, than the variations in the position which the tract as a whole assumes. It is with the characteristics of these fibers in the white rat that this paper is primarily concerned and we 411 THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, No. 4 MAY, 1913 412 S. WALTER KANSON hope to follow it with a comparative study of the pyramidal fibers in several different orders of mammals. For this reason no attempt will be made at this time to give a comprehensive review of the literature. Stieda (69) noticed that the pyramidal tract in the mouse occupied the posterior funiculus. Spitzka (’86) showed that this position was characteristic for the rat and the guinea-pig. These observations were confirmed on the rat by Von Lenhossék (’89) and Bechterew (’90), using the embryological method of Fleich- sig. Further confirmation was obtained through the application of the Marchi stain to the degenerating tract in the rat by Gold- stein (’04), Van der Vlort (’06) and Miss King (’10). TECHNIQUE In this investigation the pyridine-silver (modified Cajal) tech- nique (Ranson 712) was used as the principal method and the results were controlled by the use of the Weigert and the Pal- Weigert methods. For the Weigert methods some of the cords were fixed in Miiller’s fluid and others after fixation in 10 per cent formalin were mordanted either in Miiller’s fluid or in the following solutions: Primary mordant Bichromate of potassium ....... Se REE RE ee ob MAG iN anal aR 5.0 grams Hulyzorehroms -.:. se ncie See eee oe eee ee ele ee eee 2.0 grams Wiser, “ad. Wahi: og costae OR Oe rns ene re 100.0 ec. Nic etate! Col eC Opper.s: ate bie oe nee ne Miche uc oe eer 5.0 grams Acetic acid) (Bb) per Gent) «skin eergers whe soos Se Clee 5.0 grams Bil wonchronn # eaied < wt" s. cnireacerigt ce haere ere pee ead Be ae 2.5 grams WiBGOT ROG SRE AAN coc, visce.cia eos Sane a TAME aeRO ote eis Paseo er ees 100.0 ce. The usual staining and differentiating solutions were employed and paraffin as well as celloidin sections were utilized. An effort was made to use as many different combinations as possible, and in this way to exclude the possibility that the characteristic staining of the pyramidal tracts was due to theparticular modifica- tion of the method employed. FASCICULUS CEREBRO-SPINALIS IN THE RAT 413 The pyramidal fasciculi in the white rat take a very light stain with the Weigert methods, appearing under low magnification as grayish blue areas clearly marked off from the remainder of the white substance which stains a deep blue. On the other hand, the pyridine-silver technique causes these tracts to stand out from the rest of the cord because of the dark brown color which they assume. Since the remainder of the white substance stains a very light brown, the contrast is striking and could be equalled only by the most fortunate Marchi preparations. This contrast is equally evident in the decussation and .after the pyramidal tracts have assumed their position on the ventral surface of the medulla. Since nowhere in the literature are to be found altogether sat- isfactory figures and descriptions of the position and shape of the pyramidal tracts at different levels of the rat’s medulla and spinal cord, it seems desirable again to go over these purely topo- graphical features before taking up the finer structure. TOPOGRAPHY The changes in shape, size, and position of the tract at various levels can best be described in connection with figures 1 to 7. Figure 1 was drawn from a section through the upper end of the decussation of the pyramids. At and above this level in the medulla the pyramids are situated on either side of the anterior median suleus, but do not project ventrally as they doin the human brain. Fibers can be seen detaching themselves from the pyr- amids and running backwards to decussate as small bundles, or as individual fibers. On reaching the gray substance they spread our rather diffusely in the form of small branching bundles, many of whose fibers end within the medulla at the level of their decussation. Where the decussation is at its height (fig.2) the crossing bundles are of large size. They run backward at some distance from the central canal, and are gathered together on the dorsal surface of the gray substance into two large fasciculi. These are at first some distance apart, but approach each other in the lower part of the medulla. Figure 3 represents the lowest level of the 414 S. WALTER RANSON With the exception of figures 5 and 9, which are from Pal-Weigert preparations, the drawings were made from pyridine-silver preparations. made with a Leitz microscope. All drawings were Fig. 1 Medulla oblongata, upper end of decussation of the pyramids. Ocu. 0, Obj. 3. Fig. 2 Medulla oblongata, middle of the decussation of the pyramids. Ocu. 0, Obj. 3. decussation. The pyramids have disappeared from the ventral surface of the medulla; at a some of the lowest decussating fibers are indicated. The two large pyramidal fasciculi lie near the posterior median septum. ; The pyramidal decussation differs from that in man, in that the fibers go over into the posterior instead of the lateral funiculus, FASCICULUS CEREBRO-SPINALIS IN THE RAT 415 and in that the decussation in the rat is complete. No pyramidal fibers run directly down on the same side into the anterior funicu- lus of the cord. In the cervical region of the spinal cord (fig. 4, c. 7) these tracts occupy the ventral portion of the posterior funiculi and are closely Fig. 3 Medulla oblongata, lower end of the decussation of the pyramids; a, lowest decussating fibers. Ocu. 0, Obj. 3. Fig. 4 Seventh cervical segment of the spinal cord. Ocu. 0, Obj. 3. approximated to each other and to the curved surface of the culumna posterior. Since the medial and posterior surfaces of of the bundle are almost at right angles to each other, the shape of the tract, as seen in sections through this level of the cord, is that of one-fourth of acircle. There is not as much intermingling 416 S. WALTER RANSON of the pyramidal with surrounding fibers as one sees in the human cord. In the rat the tract stands out as sharply outlined in the preparations as it is in the drawings. In the upper thoracic region the bundle changes its shape somewhat, since the posterior surface forms an acute angle with the medial surface; and the area occupied by the tract in sections of this part of the cord has the shape of an eighth part of a circle. Figure 5 was drawn from a Pal-Weigert preparation taken at Fig. 5 Fourth thoracic segment of the spinal cord. Ocu. 3, Obj. 3. about the level of the fourth thoracic segment and shows the tract clearly outlined by its fainter staining from the remainder of the white substance. | In the mid thoracic segments the bundle becomes rounded or oval in outline, and in the lower thoracic segments (7.12) it spreads out laterally along the posterior surface of the gray sub- stance (fig. 6). In the upper lumbar region the outline of the tract is no longer so sharply indicated, and the tendency to spread FASCICULUS CEREBRO-SPINALIS IN THE RAT 417 out laterally is more pronounced. In the lower lumbar segments (fig. 7, L. 5) the fibers are diffusely scattered through the ventral part of the posterior funiculus and the tract has lost entirely its definite outline. Fig. 6 Twelfth thoracic segment of the spinal cord. Ocu. 0, Obj. 3. Fig. 7 Fifth lumbar segment of the spinal cord. Ocu. 0, Obj. 3. There is a progressive decrease in the size of the pyramidal tract as it runs caudad through the posterior funiculus of the spinal cord. This is seen on comparing the seventh cervical (fig. 4) with the twelfth thoracic segment (fig. 6). 418 S. WALTER RANSON STRUCTURE We turn now from the consideration of the tract as a whole to the characteristics of the individual fibers and shall learn why the tract stains so intensely with the pyridine-silver technique and so faintly with the Weigert methods. In _ pyridine-silver preparations of the spinal cord all the axons are stained; the larger ones are yellow, while the smaller ones are dark brown or black. The other elements of the white substance (such as myelin sheaths, neuroglia and blood vessels) are stained faintly or nor at all. Nearly all of the axons in the pyramidal fasciculus (fig. 8, a) are very small and these closely packed dark brown axons: give the characteristic brown color to the fasciculus as a whole. This contrasts sharply with the structure seen in the remainder of the white substance of the cord (fig. 8, b) where the large, ight yellow axons, surrounded by thick unstained rings of myelin give rise to a lighter color and a more open structure. There are a few medium-sized axons in the pyramidal tract and a few of the very fine ones in the other fasciculi of the cord. Figures 8 and 9 were taken from the fourth thoracic segment of the spinal cord at the border of the pyramidal fasciculus. As has been said, the pyrimidal tract takes a light grayish blue stain in Weigert preparations (fig. 5). It contains many very fine medullated fibers and a few of medium size (fig. 9,a). The myelin sheaths of the pyramidal axons are thinner than the sheaths on axons of the same size in other regions of the cord. Some are so thin and faintly stained that they are just recogniz- able. The medullated fibers do not occupy all the space in the tract but are separated from each other by unstained spaces. When we compare the Weigert and the pyridine-silver prepara- tions of the same level of the cord we see that the axons in the pyramidal fasciculus (fig. 8,a) are much more numerous than the myelin sheaths (fig. 9,4) and that the axons are more closely packed together. In any given section, therefore, many of the axons are without myelin sheaths. This is susceptible of two interpretations: either many of the pyramidal fibers are entirely non-medullated; or the myelin sheaths of the pyramidal fibers are FASCICULUS CEREBRO-SPINALIS IN THE RAT 419 interrupted, medullated and non-medullated stretches succeed- ing each other along the course of the same fiber. In any case, the medullation of the pyramidal tract in the white rat is incom- plete, and such sheaths as are present are very thin. It is not possible to draw the same sharp line between entirely non-med- ullated and fully medullated fibers that can be drawn in the spinal nerves. se aerts hid a. ee e eA SS Bie’ OSHA “2 we = oe ce eto ert Q a m~ Cy ee Bek ee Op (SPOS TTD 5 Pao ad = HA 9, e o) = Lh Wer ee “22 8 oth) QQ a-e FNM NS ee O78 A) b 0 la Bre sho, oe inas S,! Reese” Be vs, eo So Sew eh 2 we f) ° s O3.°e yo» tS ax ¢ SS 5Q 009, @o%& £08 I SERS ¢ 5 g. C6 00% 8 G20 ao0 S SEO Sp. Sba 0 Io 0. 6 a °, Go ce) : sod: 90 "DD a © ego 2 oe AT Rd S 8 9 Fig. 8 An area from the fourth thoracic segment of the spinal cord at the boundary between the pyramidal, a, and the cuneate, 6, fasciculi; only axons are shown. Ocu. 3, Obj. 2 mm. ; Fig. 9 An area from the fourth thoracic segment of the spinal cord at the boundary between the pyramidal, a, and the cuneate, b, fasciculi; the myelin sheaths are shown. Ocu. 3, Obj. 2mm. el ae ne mill. The same results are given by both the old Weigert and the Pal-Weigert methods. Care has been taken not to decolorize the smallest myelin sheaths. To avoid this, sections8 to 10 » thick were used, and the decolorization was stopped just short of com- pletion, leaving a diffuse light blue tint in the background. In these thin preparations it was possible to see dark blue myelin sheaths clearly outlined against the lighter background. The number of sheaths seen in this way corresponded with the num- ber seen in the more fully differentiated preparations. When a well differentiated preparation is stained with acid fuchsin a 420 S. WALTER RANSON counter-stain of the axons is obtained. The pyramidal tract stains intensely with the fuchsin because of the predominance of axon substance in its composition. Non-medullated fibers are also found in other parts of the white substance of the rat’s spinal cord but are much less numer- ous than in the pyramidal fasciculus. It should be added that all these observations were made on well developed adult rats, and are not to be explained by an immaturity of the individual animals employed. These observations on the character of the fibers in the pyra- midal tract of the white rat were made in connection with a search for the path within the spinal cord taken by the non-med- ullated fibers of the dorsal roots (Ranson 712). It is conceivable that they might run into the ventral portion of the posterior funiculus and ascend in the region occupied by the pyramidal tract; and since the number of non-medullated fibers from the dorsal roots is very considerable, such ascending fibers might represent all of the non-medullated fibers seen in this part of the cord. That is to say, the tract described in the first part of this paper might be a mixed one consisting of descending med- ullated fibers from the motor cortex and ascending non-medul- lated fibers from the dorsal roots. In order to rule out this possibility, the following experiment was performed on adult albino rats. Under aseptic precautions the sciatic nerve was exposed in the upper part of the thigh, grasped with artery forceps and torn out of the pelvis. Five experiments were made. In one case two dorsal roots and their ganglia came away with the sciatic, in the remaining four only one root and ganglion. Each animal was killed after from twenty-four to twenty-eight days. No attempt was made at the autopsy to determine which of the roots associated with the sciatic was torn away in the operation. Pyridine-silver preparations were made of the lumboscaral portion of each of these cords. A varying degree of degeneration was seen in the last lumbar segments, depending upon the amount of damage done to the ganglia and roots when the sciatic was torn out. But in each case a very definite degenerated area could be seen, from which most of the FASCICULUS CEREBRO-SPINALIS IN THE RAT 421 axons had disappeared. Figure 10 illustrates the appearances seen in these sections. It will be observed that the posterior funiculus on the left side of the drawing is considerably smaller than that on the right and contains a degenerated area (a) reach- ing from the surface of the cord to the interval between the two pyramidal fasiculi: The area occupied by large undegenerated medullated fibers on the left side (b) is about one-half that on the right. But there is no clearly marked decrease in the size fo oe Aa tharine Win (112. 10 Fig10 Part of asection through the lumbar portion of the spinal cord showing degeneration, a, in the cuneate fasciculus. Undegenerated portion of the cuneate fasciculus, b; gray substance c. Ocu. 3, Obj. 3. of the pyramidal fasciculus nor any noticeable degeneration of the axons within its area. In order to compare more accurately the areas occupied by the pyramidal fasciculi on the normal and operated sides, these areas were roughly computed in sixteen successive sections. This was done by tracing the outlines of the tracts with the camera lucida on millimeter paper, and determining the number of square millimeters covered by the areas thus projected. It was found that the tract on the operated side was3 per cent smaller than that 422 S. WALTER RANSON on the normal side, a decrease which could be accounted for by the presence of a few dorsal root fibers within the area of the tract. Since the tract decreased so little in size and since there were no other evidences of degeneration within its territory, it is obvious that the non-medullatd fibers which it contains do not belong to another system arising in the dorsal root ganglia. These experiments also serve to emphasize the sharpness with which the regions occupied by the pyramidal tracts are limited in the white rat. Watson (’03) noticed that in the Pal-Weigert preparations of the spinal cord of the adult albino rat the pyramidal fasciculus was only slightly stained and he attributed this to a supposedly different chemical composition of the myelin in the sheaths of these fibers. Miss King (10) states that, when compared with the Marchi preparations of the pyramidal fasciculus in the rabbit, cat and dog, the Marchi preparations of this tract in the rat reveal a striking paucity of fibers ‘‘so that in this animal the so- called primary motor path is probably only of secondary impor- tance.’”’ In view of the incomplete medullation of the pyramidal tract in the rat it is easy to understand Miss King’s results. Although there is an abundance of axons there are few well med- ullated fibers, such as would respond readily to the Marchi stain. Just how far the incomplete medullation of this tract is an indi- cation of an incomplete development of its function is a matter which it would be very difficult to decide. Attention has been called to the peculiar light staining of this fasciculus in Weigert preparations of the spinal cord of animals belonging to widely separated groups. Ziehen (’99 and ’00) mentions it as occurring in the pseudochirus, the sheep and therat. Driseke (’04) observed that in the mole the pyramidal fibers lose their myelin sheaths as they pass from the medulla into the anterior funiculus of the spinal cord, into which they go without decussation. Here they form a medially placed oval field somewhat ventral to the ante- rior commissure. This oval area is very faintly stained in Weigert preparations and is almost devoid of medullated fibers. Bischoff (00) states that in the hedgehog the pyramidal fibers are so fine and possess so delicate a myelin sheath that the Marchi stain FASCICULUS CEREBRO-SPINALIS IN THE RAT 423 does not give good results. He could follow only a few degen- erating fibers into the spinal cord where they lay in the homolat- eral anterior funiculus. They could not be traced beyond the upper cervical segments. These findings seem to indicate a con- dition in the hedgehog similar to that found by Driseke in the mole. It would seem, therefore, that the incomplete medullation of the pyramidal tract in the rat is not a characteristic peculiar to these animals but is related to similar conditions in‘at least some marsupials (pseudochirus), some species of insectivora (mole, hedgehog), some other rodents (guinea-pig) and some ungulates (sheep). Since, however, the observations on these other animals were confined to myelin sheath stains it seems desirable to make a comparative study of the question with an axon stain. Such an investigation is now under way. In man, medullation of the pyramidal tracts begins shortly before birth and is not completed until the second year. It is obvious that this tardy medullation is rendered more significant in the light of these facts concerning the condition of this tract in some of the lower animals. BIBLIOGRAPHY BEcHTEREW, W., 1890 Ueber die verschiedenen Lagen und Dimensionen der Pyramidenbahnen beim Menschen und den Thieren. Neurol. Cen- tralbl., Bd. 9, S. 738. Biscuorr, E. 1900 Beitrag zur Anatomie des Igelgehirnes. Anat. Anz., Bd. 18, p. 348. Drisexe, J. 1904 Zur Kenntnis des Riickenmarks und der Pyramidenbahnen von Talpa europaea. Monatschr. f. Psy. u. Neurol., Bd. 15, p. 401. GoupsTEIN, G. 1904 Zur vergleichenden Anatomie der Pyramidenbahn. Anat. Anz., Bd. 24, p. 451. Kina, Jessie L. 1910 The cortico-spinal tract of the rat. Anat. Rec., vol. 4) p. 245. von Lennossix, M. 1889 Uber die Pyramidenbahnen im Riichenmarke einiger Siugetiere. Anat. Anz., Bd. 4, 8. 208. Ranson, S.W. 1912 The structure of the spinal ganglia and of the spinal nerves. Jour. Comp. Neur., vol. 20, p. 159. 424 S. WALTER RANSON Srirzka, EK. C. 1886 The comparative anatomy of the pyramid tract. Jour. Comp. Medicine, vol. 7, p. 1. Strepa, L. 1869 Studien itiber das centrale Nervensystem der Végel und Saugethiere. Zeitschr. f. wissensch. Zoologie, Bd. 19, 8. 1. VAN DER VioRT, 1906 Ueber den Verlauf der Pyramidenbahn bei niederen Saiugetieren. Anat. Anz., Bd. 29, p. 113. Watson, J. B. 1903 Animal education. Chicago, 1903. ZIEHEN, TH. 1899 Zur vergleichenden Anatomie der Pyramidenbahn. Anat. Anz., Bd. 16, p. 446. 1900 Ueber die Pyramidenkreuzung des Schafes. Anat. Anz., Bd. Ligap- DOK. ON THE DEVELOPMENT OF THE MEMBRANA TECTO- RIA WITH REFERENCE TO ITS STRUCTURE AND ATTACHMENTS C. W. PRENTISS The Anatomical Laboratory of the Northwestern University Medical School, Chicago FOURTEEN FIGURES For more than half a century various investigators have studied the structure of the cochlea with conflicting results. Compara- tively recently Kishi (’07) and Shambaugh (’07) have championed the view originally held by Retzius (’84), that the membrana tectoria remains attached to the organ of Corti. They maintain moreover that the membrana is the logical structure through which sounds are transmitted to the auditory cells, and that it acts as aresonator. This function Von Helmholtz was the first to ascribe to the rods of Corti and later with Hensen to the fibers of the basilar membrane. Hardesty (’08) denies the existence of an attachment between the membrana and the spiral organ, yet maintains that through the medium of the membrane sound vibrations are transmitted to the auditory hairs. A voluminous literature has been written dealing with the physiology of an organ the structure of which is inadequately known. To ascribe a definite function to the membrana tectoria we must first know with absolute certainty its structure and attachments. No physicist will accept as an important organ of hearing a membrane of indefinite structure and with no fixed position with reference to the spiral organ itself. For such a floating membrane, as we shall show later on, may readily change its position and relations to the auditory cells, and would certainly interfere with and interrupt the audi- tory function. rn to ol 426 Cc. W. PRENTISS From the physiological standpoint it is then necessary to answer two anatomical questions before we may assign the mem- brana a logical réle in the processes of audition: (1) Has the membrana any definite and peculiar structure which may adapt it to the transmission of sound vibrations? (2) Is the membrana so attached as to be constantly in contact with the hair cells of the spiral organ? We hold that as yet these questions have received no adequate answer. The membrana is described by nearly all of those who have investigated it, as being an elastic cuticular structure containing within its interstices a more or less fluid matrix. This cuticular membrane has been variously interpreted as formed of aggluti- nated cilia or hairs (Ayers 792); as lamellar (Shambaugh ’07); as a reticulum (Retzius ’84); as a coagulum of the endolymph (Czinner and Hammerschlag ’98); as a fibrous feltwork embedded in a gelatinous matrix (Hardesty ’08); as composed of fibers and cuticular layers (Held ’09). As to its attachments there is a division of opinion, some holding that it is attached to the spiral organ (Retzius ’84, Coyne et Cannieu ’95, Kishi ’07, Shambaugh ’07) while this is denied by others—more recently by Rickenbacker (’01), Hardesty (’08) and Held (’09). The classic figures given in textbooks of anatomy and histology (fig. 1) show it as a lamellated membrane attached to the labium vestibulare and extending outward over the internal spiral sulcus and the organ of Corti. Its outer edge thus floats free in the endolymph and the lamellae are shown parallel to the ends of the hair cells. The textbooks usually state that it takes its origin from the limbus spiralis and hence must grow by the development of new lamellae from beneath. The conclusions of those who have worked on the development of the membrana tectoria are as contradictory as are those who have interpreted its structure. Kdlliker (’61) originally de- scribed the membrana tectoria as a finely striated membrane arising from the columnar epithelial cells of the basal wall of the ductus cochlearis. Hensen (’63), Retzius (’84), Pritchard (’78), Schwalbe (’87) and others agree as to its cuticular origin. Czinner « DEVELOPMENT OF THE MEMBRANA TECTORIA 427 and Hammerschlag (’98) assert that it arises independently as a coagulum or concretion of the endolymph, and later becomes attached to the epithelium. Ayers (92) maintained that the fused hairs of the auditory cells form the membrana tectoria and that their agglutinated tips later fuse to the labium vestibulare, while Béttcher (70) asserts that it is formed from hairs arising from the epithelial cells of the cochlear duct. Coyne et Cannieu (95) found that the membrane was attached to the organ of Corti or had been torn away from it and that it shows a lamellar or reticular structure according as it is sectioned through the lindbus membrana tectoria outer hair-cells — ae nerve fibres inner rod vas basilar outer cells of Deiters spirale membrane rod Fig. 1 Semi-diagrammatic representation of the organ of Corti and adjacent structures (Merkle-Henle). axis (modiolus) of the cochlea or in a plane perpendicular to this. There are thickenings at the angles of the reticulum and these thickenings give to the membrane the striated appearance which is seen in other sections. Rickenbacher (’01) after studying five stages in the develop- ment of the cochlea of the guinea-pig concludes that there are two epithelial ridges (wuelste) in the floor of the cochlear duct. The inner axial ridge is the greater and its cells give rise, first, to the major part of the membrana tectoria as a cuticular secre- tion which increases in thickness outwardly by the addition of new lamellae. Later from these cells are formed the epithelium THE AMERICAN JOURNAL OF ANATOMY, VOL, 14, NO. 4 428 C. W. PRENTISS of the labium vestibulare, of the spiral suleus and the inner portion of the spiral organ. The lesser or outer ridge gives rise to the outer portion of the membrana tectoria, and later forms the outer portion of the spiral organ. In the new-born guinea- pig the tectorial membrane loses its connection with the organ of Corti, probably owing to a dissolution of a portion of the membrane. The membrane makes its appearance before the differentiation of the organ of Corti or of the hair cells. In structure it is a cuticular reticulum which later becomes swollen, convex above and is detached from the spiral organ by the secretion of endolymph beneath it. No cilia or hairs were ob- served by Rickenbacher until after the differentiation of the organ of Corti, and then only the hairs of the auditory cells appeared. He does not state definitely just how the cuticular reticulum of the membrane arises nor does he account for the striated or lamellar structure which is characteristic in ordinary preparations and which he figures. Hardesty (’08) in restudying the development of the membrana tectoria finds it first in embryos of 3 cm. as a “‘cuticular film of appreciable thickness and decided fibrous character.’’ Of the two epithelial thickenings in the basal epithelium the inner only takes part in forming the membrana tectoria, the outer giving rise only to the spiral organ (of Corti). ‘‘Not till pigs of about 14 em. do any preparations show evidences of differentiation of the cells of the lesser thickening into what will become the organ of Corti,” says Hardesty. He thus agrees with Rickenbacher that the mem- brana is quite well formed before the hairs of the auditory cells appear, thus proving false the conclusions of Ayers. As he con- tends that the membrana does not develop over the spiral organ (though his figure 10 does not bear out this contention) Hardesty must account for its later position over and extending to the outer side of the hair cells. This he does by maintaining that owing to the retrogression of the cells which originally fill the spiral sulcus, there is an inward displacement of the spiral organ which thus causes the outer portion of the membrana to rest above and beyond it. Just how this can take place without a shortening of the basal membrane is not stated, nor do his meas- DEVELOPMENT OF THE MEMBRANA TECTORIA 429 urements show conclusively that the pillar cells have actually approached the inner angle of the cochlea a sufficient distance to warrant the change in position of the membrana tectoria. This point will be taken up in describing our own preparations. Hardesty describes an accessory tectorial membrane lying beneath the tectorial membrane proper and extending from near its outer edge to Hensen’s stripe. It is composed of two sets of fibers crossing at an acute angle. In his figures these fibers form a network with diamond shaped meshes. Hardesty does not state how this accessory tectorial membrane is developed. Hensen’s stripe, a line which has been described as extending lengthwise along the underside of the tectorial membrane, Hardesty regards as due to the intercrossing ends of the fibers composing the membrane. He states that its position corresponds to the line of enclasped phalanges of the pillars. Hence the stripe of Hensen should lie between the inner and outer hair cells. Hardesty believes that it has an embryological significance: ‘‘Hensen’s stripe seems to be the expression of the period at which the retrogression of the epithelium began. It also represents the line along which the thick, outer edge of thickening was last attached and along which growth was last contributed to the membrane.”’ Held (09) has made a detailed restudy of the development of the organ of Corti and the membrana tectoria in the ear of the guinea-pig, rabbit, pigeon and chick. He finds that the membrana tectoria is developed as cuticular fibers by the cells of the basal epithelium of the cochlear duct. An outer cuticular layer is first formed over the greater epithelial thickening; later growth con- sists in the secretion of the fibers by the cells of both thickenings, the hair cells alone taking no part in their development. Thus he holds that the membrana tectoria is developed in situ over the organ of Corti. Owing to the later elongation of its cells the organ of Corti shifts its position inward (axially) but this shifting is not extensive enough to account for the position of the membrana tectoria, which overlies and may project beyond the cells of. the organ. In the adult fowl Held found that the membrana remains attached to the supporting cells of the sensory 430 Cc. W. PRENTISS organ, but believes that in adult mammalia its attachment to the cells of the organ of Corti is lost. Because of these contrary and diverse conclusions which the literature in regard to the development and structure of the membrana tectoria contains, and because of its importance to - the physiology of audition it was determined to make a restudy of its development in pig embryos and of its structure in man. The present paper includes my work on the development only. METHODS Experimenting with fixing fluids it was found that formalin and Zenker’s fluid preserved the membrana well but failed to bring out sharply its cuticular structure. Osmic acid of 2 per cent and Vom Rath’s osmic-picric-acetic mixture was used with success in the later stages as the fixation was good and the brown- ing of the cuticulum by the osmic acid made its structure more clear. In the younger stages the whole head of the embryo was fixed. In the stages approaching full term the bony labyrinth was shelled out whole, and, after the stapes had been carefully removed, was immersed in the fixative two to three days. After fixation and hardening the decalcification of the older stages was completed in 80 per cent alcohol plus 5 per cent nitric acid. They were then embedded in celloidin or paraffine and cut in planes parallel and perpendicular to the modiolus of the cochleae. Preparations were thus made from pig fetuses measuring 4, 5.5, 7.5, 8.5, 18, 15, 18.5 and 20 cm. and these were compared with sections from full term fetuses. It was found that sections mounted in balsam were not favorable for a study of the membrana because its cuticular framework is of about the same refractive index as this mounting medium. Celloidin sections were therefore mounted in water, and while only temporary preparations could be made in this way, this method was of great value in determining the structure of the membrane when unstained. With sufficiently thin sections an oil immersion objective could be employed. No special staining methods were used, the browning of osmic acid DEVELOPMENT OF THE MEMBRANA TECTORIA 431 being more effectual than any stain. Nuclei were demonstrated with haematoxylin and for counter stains eosin, orange G, and acid fuchsin were employed. DESCRIPTION OF STAGES Aside from the thickening of the basal epithelium, stages up to 4 mm. show no important changes. Fig. 2 Section through the second turn (fig. 13) of cochlear duct from a 5.5 em. fetus, showing basal epithelium and origin of membrana tectoria; inner or axial side to left; m.vest., membrana vestibularis; m.tect., membrana tectoria; gang.sp., spiral ganglion; ¢.sp., spiral tunnel. Oc. 4, Obj. 4. 5.5 cm. stage. Here we have the basal epithelium composed of pseudo-stratified columnar cells (fig. 2). The nuclei of the high columnar cells show a division into an inner and outer group which correspond to the greater and lesser epithelial thickenings of Rickenbacher. A section through the cochlea of a somewhat later stage shows the topography of 432 Cc. W. PRENTISS the spiral organ (fig. 13). The cochlear duct of the pig makes about 3.5 turns and hence a section through the modiolus shows four turns on the left and three on the right in figure 18. Figure 2 represents the basal half of the turn lettered (2) in figure 13. Of the two groups of cells which we have noted above the larger group forms the inner (axial) two-thirds of the basal wall. The nuclei of these cells are arranged in from three to six layers. They are separated from the outer group of cells by a cytoplasmic area free from nuclei. Between two cells of this area a vertical space is seen extending from the summits of the cells half way through the epithelium. This space may be due to shrinkage but represents the position of the spiral tunnel or tunnel of Corti in later stages. The outer cell group (lesser thickening of Ricken- bacher) forms the outer third of the basal wall of the cochlear duct. It will eventually give rise to that part of the spiral organ - which lies external to the spiral tunnel (fig. 1). From the inner epithelial cell group (greater epithelial thickening) will develop the epithelium of the labium vestibulare, of the internal spiral sulcus and that portion of the spiral organ lying internal (axial) to the tunnel. Extending over the free ends of the cells of the greater epithelial thickening may be seen a cuticular membrane which is attached between the cells by delicate threads. This cuticular membrane is the anlage of the membrana tectoria, which thus makes its appearance before the hair cells of the spiral organ are differentiated. At this stage the mesenchyma about the cochlear duct is dense and the scalae have not yet appeared. The nerve fibers of the spiral ganglion may be seen entering the epithelium internal to the organ of Corti. 8.5 cm. stage. In the second turn of the cochlea at this stage (fig. 3 and fig. 13):a space representing the spiral tunnel extends nearly through the thickness of the epithelium. The cells on each side of the tunnel are differentiating the pillars of Corti and a single inner and three outer hair cells are conspicuous. By the rapid division and elongation of the cells of the greater epi- thelial thickening the epithelial wall has been bent basalwards forming a concavity above and a convexity below. The concavity is the first trace of the internal spiral sulcus. The nuclei of the DEVELOPMENT OF THE MEMBRANA TECTORIA 433 greater cell thickening show not so many layers as in the pre- vious stage but the cells are longer than the pillar cells of the spiral organ. Near the inner angle of the cochlear duct the membrana tectoria has increased very little in thickness and still forms a thin cuticular layer over the epithelial cells. Externally the membrana now extends beyond the outer hair cells of the spiral organ at which point a thin cuticle is just being formed. Over the spiral sulcus the growth of the membrana has been most rapid and here it is thickest. It appears to be composed “bff ie THs WAM) 19% 4 8) gle 2.010048 80 Fie a 6/9889 //, 0) ay ae A as ed | Bib ites Y- $e he T) ONS Pod, Fig. 3. Section through the second spiral of the cochlear duct from a fetus 8.5 cm. long, showing the basal half of the cochlear duct and a portion of the scala tympani; h.c., hair cells of spiral organ; 7.ep.c., inner epithelial thickening; 0.ep.c., outer epithelial thickening; sc.tymp., scale tympani; other lettering as in figure 2. Oc. 4, Obj. 4, t. 1. 160. of numerous parallel fibers or lamellae which are attached to the epithelium between the cells. When traced upwards away from the cells the lamellae converge and curving inwardly are con- tinuous with the thin plate-like inner portion of the membrane which overlies the labium vestibulare. The appearance of the tectorial membrane at this stage has been explained correctly, we believe, by Hardesty (08). After a cuticular layer has been formed as in figure 2, the cells internal to the sulcus spiralis secrete very slowly or cease altogether. The 434 C. W. PRENTISS other cells which are forming the membrana continue to secrete actively. At the same time these cells by growth and multipli- cation increase the width of the basal epithelium, carrying the spiral organ outwards. Thus the distance from the inner angle of the cochlear duct to the spiral tunnel is increased. In the second spiral of the 5.5 em. stage as in figure 2 this distance is 140». In the 8.5 em. stage the same distance is about 280 u. Cells near the pillars of Corti which are secreting the membrane may thus be carried outward approximately 140 4, while that part of the membrane first formed does not grow. As the so- called ‘lamellae’ are secreted at the ends of the cells and the cells are shifted outward as the lamellae lengthen, naturally the bases of the lamellae will also be carried outward while their tips remain stationary. The inward trend of the lamellae from base to tip is thus satisfactorily accounted for. It may be well to emphasize here the fact that the greater epithelial thickening gives rise not only to the epithelium of the labium vestibulare and of the spiral sulcus but also to the inner axial half of the spiral organ, including the inner supporting cells, and possibly the inner hair cells and inner pillars. This is in agreement with the results of Coyne et Cannieu (’95) and Rickenbacher (’01) Van der Stricht and Held (’09). Hardesty states that ‘“‘the lesser thickening is the first indication of the differentiation of the organ of Corti while the cells of the greater give origin to the tectorial membrane . . . . and the low indifferent cells lining the spiral suleus.’”’ This misinterpretation is important as it partly accounts for his later statement that the membrana is not derived from the cells of the spiral organ. The next question to decide is the true structure of the mem- brana tectoria. Is the membrane composed of lamellae or hairs or fibers or is it a reticulum? If it is formed at the ends of the cells just how is it developed there? These points were decided by a study of later stages, the cochleae of a 13 cm. fetus prov- ing most favorable material. In the various cochleae which were examined it was found that differentiation begins in the basal turn and is much less advanced in the upper turns. Thus DEVELOPMENT OF THE MEMBRANA TECTORIA 435 in the 5.5 em. stage the membrana tectoria was not yet devel- oped in the upper turn though it had appeared in the second turn. The upper spiral of the 8.5 cm. stage was only slightly advanced in development beyond that of the basal turn of the ' previous stage. 13 cm. stage. The upper coil in this stage showed but little more differentiation of the tectorial membrane than figure 3. In the second spiral, however, a marked difference may be seen (fig. 4). A fibrous basement membrane is stretched beneath the epithelium, extending between the limbus spiralis and the spiral Fig. 4 Section through the basal portion of the second turn of the cochlear duct from a 13 em. fetus; m.bas., membrana basilaris; limb.sp., spiral limbus. X shows point at which later labial teeth appear separating labium from the sulcus; *marks portion of field similar to that shown in figure5. Oc. 4, Obj. 4, t. 1. 190. ligament. The epithelium itself shows a greater thickening in the region of the future sulcus spiralis, the cells being more elongate and clearer. At the axial side they show but one row of nuclei. At X these cells are sharply marked off from the epithelial cells of the labium vestibulare, the latter cells forming the so-called teeth of Huschke. The sulcus spiralis is somewhat deeper than in the preceding stage and the outer supporting cells of the spiral organ are longer and more sharply differen- tiated from the single layer of cubical cells external to them. The membrana tectoria is larger and extends from the inner angle of the cochlea duct to well beyond the spiral organ externally. 436 C. W. PRENTISS Over the labium vestibulare it forms a thin nearly structureless cuticular layer which becomes thicker and shows lamellae over the labial teeth. In this region it is detached from some of the epithelial cells, a condition due to shrinkage. In the region of the future sulcus spiralis and over the inner portion of the spiral organ the membrana tectoria appears com-_ posed of delicate parallel plates which have the appearance of hairs or fibers in section, and have so been interpreted by some investigators. These plates may often be traced between the cells or about their ends. They are separated by spaces which correspond frequently to the width of the cells at the surface of the epithelium. As one follows the plates away from the epi- thelium the spaces become smaller and the plates or lamellae approach each other until the membrana has the appearance of a solid structure with fine parallel striations; striations which, as we have seen, converge towards the inner angle of the cochlear duct. The relation of the plates or lamellae to the cells lining the future sulcus spiralis is shown in figure 5. Using an oil immer- sion objective the lines were seen as sharply as in a diagram, many passing between the cells and thus taking their origin as an inter-cellular secretion. The thicker lines undoubtedly repre- sent two plates agglutinated. Thus far we have shown, conclusively it seems to us, that the membrana tectoria takes its origin partly from cells which in the adult line the spiral sulcus and partly from the inner supporting cells of the spiral organ; and that the cuticular plates are not like hairs or cilia in their development, as they may be traced between the cells. The next question is whether the outer cells of the spiral organ takes part in the formation of the membrana. Figure 6 shows the relation of the membrana tectoria to the cells of the spiral organ. At this stage the membrana is composed of a thin cuticular plate attached between | the ends of the cells by what are apparently delicate threads. Internally (axially) the membrane is thicker and shows converg- ing striae. Externally the membrana extends well beyond the cells of the spiral organ. The hairs of the two outer auditory cells were apparently attached to the outer surface wall of the DEVELOPMENT OF THE MEMBRANA TECTORIA 437 membrana. Except in so far as they are enclosed therein the hairs have nothing to do with the development of the tectorial membrane. ACTA ice Fig.5 Portion of basal epithelium indicated by * in figure 4, showing the struc- tural relation of the membrana tectoria to the columnar cells; m.tect., membrana tectoria; X, threadlike lamellae originating between two epithelial cells; col.ep., columnar cells of inner group; m.bas., basilar membrane. Oc. 2, 2 mm. Obj., Go,1:-L60: Fig. 6 Drawing showing the membrana tectoria developing over the hair cells of the spiral organ (marked h.c. in fig. 4); 7.h.c., inner hair cell; 0.h.c., outer hair cell; t.sp., spiral tunnel. Ob. 4, 2mm. Obj. 438 C. W. PRENTISS In describing a 14 em. stage Hardesty says: ‘‘ Up to this stage, the membrane never overlaps the lesser thickening and in confirma- tion of the statement of Rickenbacher it must be said that at no stage is there good reason to assume that the cells giving rise to the organ of Corti ever have anything to do with its development.’} Rickenbacher in his summary states that ‘‘ Die Cortische mem- bran ist somit doppelten Ursprungs: Die innere Zone ist die primire welche von grossen Epithelialwulst abgeschieden wird. Die schmale Randzone ist eine sekundire Bildung, welche an dem kleinen Epithelialwulst abgesondert wird.” As Ricken- bacher states that the outer portion of the spiral organ is devel- oped from the lesser ‘Epithelial-wulst’ and the inner portion from a part of the greater ‘Epithelialwulst,’ the above quotation is not in accord with Hardesty’s statement. Rickenbacher’s figures (11, 12, 13) show the membrana developing over the cells of the spiral organ and attached to the hairs, so also do the figures of Held (09) and even in Hardesty’s figure 10 the mem- brana is shown projecting beyond thé outer pillar cells and attached to the inner cells of the spiral organ. On dissecting away the membrana at this stage it was found that the thin platelike zone, overlying the spiral organ, and extending beyond it was no artifact due to coagulation, but a definite structure of the same appearance and continuous with the rest of the mem- brane. The total width of this membrana was found to be equal approximately to that of the membrana in the same turn of a 18.5 cm. stage. In our descriptions we have referred heretofore to the struc- tures composing the membrana tectoria as ‘lamellae or fibers.’ From the manner in which these are attached to the cells and from horizontal sections it will be seen that such terms can not rightly be applied to them. Sections of the membrana cut through the cochlea perpendicular to the modiolus or axis show their true significance. In such a section the organ of Corti (spiral organ) and the limbus are cut at right angles to the long axes of their cells, the line of section being indicated by 2 in figure 1 [Italics mine. DEVELOPMENT OF THE MEMBRANA TECTORIA 439 5. That portion of the membrana extending over the sulcus is seen cut perpendicular to the fibers or lamellae (fig. 7). Its structure is that of a reticulum. The meshes are composed of delicate cuticular walls and at their angles are triangular or rectangular thickenings. The walls of the network are sharply defined and in unstained preparations appear highly refractive and clear. This structure can not be due to the effects of fixing reagents upon a gelatinous substance for in this case the lines of strain would not be as definite and would have a grayish, granular appearance instead of being clear and refractive as is Fig. 7 Membrana tectoria sectioned in a plane perpendicular to the axis of the cochlea, thus cutting across the ‘fibers.’ The drawing shows its reticular structure with thickenings at the angles of the meshes. From a fetus of 15 cm.; ep., epithe- lium. Oc. 2,2mm. Obj., t. 1. 160. Fig. 8 Diagram showing structure of the membrana tectoria as proved by figures 4 to 7; r., reticulum, as seen in horizontal sections; l., ‘lamellae’ or ‘fibers,’ seen in axial sections. the case with this cuticulum of the membrana tectoria. The spaces enclosed by the network correspond in form and size to cross sections of the epithelial cells and where*the membrana approaches the epithelium may be seen to correspond to the ends of the cells. The structure of the membrane is thus neither lamellar nor reticular but ‘cellular’ in the sense that honeycomb is cellular. The cuticular portion of the membrane corresponds to the waxen cells and these chambers are closed during develop- ment by the ends of the epithelial cells. There is this difference in the comparison that while the ‘cells’ of a honeycomb are nearly 440 Cc. W. PRENTISS straight and of the same diameter throughout, the chambers in the membrana taper as we go from the epithelium and curve toward the inner angle of the cochlear duct and are probably irregular in length and arrangement. My conception of the structure of the membrana based upon the preparations already described is shown diagrammatically in figure 8. The reticular structure is shown at the bases of the chambers, the thickenings at the angles of the meshes extend lengthwise of these chambers and when seen in side view, as in axial sections, they give the membrane the fibrous or striated appearance which has been so frequently described. This appearance was rightly interpreted by Coyne et Cannieu (’95). In a vertical section usually more than one layer of cuticular chambers may be seen and hence the striations appear numerous, indistinct and close together. Few investigators have made horizontal sections of the cochlea and in the adult and in late fetal stages such sections are difficult to obtain. Hardesty shows a section (fig. 9) in which at a cross sections of the ‘fibers’ are seen, and he has drawn a reticulum with thickenings at the angles. He states that the fibers seem to anastomose and appear to be connected with each other by fine collateral filaments but attributes this appearance to shrink- age and coagulation. On pages 161 to 162 he states that “The membrana is not a lamellated structure. Ever since 1869 when Bottcher teased portions of it and found them to contain fibers the fibrous structure of the membrane has been conceded by all who have studied it with reference to its structure. Sections in different planes, as made by Coyne and Cannieu (’85) [’95] and here (Fig. 9) indicate clearly its fibrous structure.’’? It is certain that Shambaugh did not concede its fibrous struc- ture, as he states that it is lamellated. On page 132 Hardesty states: “Lowenberg (’64) thought that the membrane consisted of layers one above the other; Gottstein (’72) pictured it as structureless, and many others after these have failed to compre- hend its character.’’ Rickenbacher does not figure any very definite structure nor does he account for its development. As 2 Italics mine. DEVELOPMENT OF THE MEMBRANA TECTORIA 44] to Coyne et Cannieu (’95), in describing the sections mentioned by Hardesty as confirming the fibrous character of the membrana, they say (p. 285): Cette membrane offre l’aspect d’un réseau, dont les travées serient constituées par une substance amorphe, claire et transparente. Ces travées circonscrivent des cavités polygonales diminuant d’epaisseur & mésure qu’on s’eloigne de l’organe de Corti pour se rapprocher de la protubérance de Huschke. Les cloisons de ces cavités se réunissent au niveau des angles du réseau et forment, en ce point, des espaississments sur toute la longueur de leurs bords de réunion. Ces espaississments sur des coupes radiales de la membrane se montrent sous l’aspect des stries dont nous avons déja parlé. Coyne et Cannieu thus are in agreement with my interpre- tation of the structure and on page 280 state definitely that the membrana is not composed of fibrils imbedded in a homogeneous matrix. Hardesty could not demonstrate by special stains the presence of a matrix which would hold the fibers together. His conclusions are based apparently on surface views of the mem- brana in which he saw an apparent fibrillar structure. The ends of the fibers which one may see on the under side of the mem- brana may be interpreted also as the thickenings at the angles of the reticulum shown by Coyne et Cannieu and myself and as drawn by Hardesty himself in figure 9. It is improbable that this structure can be due to shrinkage and coagulation, for the walls of the meshes are sharply defined, clear and refractive, the size of the meshes corresponds to the size of the cells in trans- verse section and the network may be seen attached between cells of the spiral organ when studied in serial sections. The accessory tectorial membrane which Hardesty describes as composed of two sets of obliquely crossing fibers he figures as a reticulum with ‘diamond’ shaped meshes. Its probable struc- ture is that of a reticulum and it may be explained as a thin layer of the membrana tectoria which was left adherent to the spiral organ and later was torn away. It probably represents the reticular membrane or lamina reticularis of the spiral organ which Coyne et Cannieu interpret as a portion of the membrana tec- toria which has remained attached to the cells of the spiral organ. Horizontal sections also explain why the membrana, or portions 442 Cc. W. PRENTISS of it, have been described by some as having a reticular struc- ture. Held (’09) figures the membrana as arising from the ends of the supporting cells of the basal epithelium in the form of parallel fibers. Yet he does not show these fibers as continuous with the cytoplasm of the cells, like the hairs of the auditory cells, nor did he study horizontal sections through the membrana. To sum up the development of the membrana previous to fetuses of 15 cm., we may say that it is a cuticular organ with a definite though irregularly chambered structure which ws secreted between, and at the ends of the cells composing the basal epithelium of the cochlea. Both the greater and lesser epithelial thickenings take part in its development, its outer zone arising between the cells of the spiral organ. It appears first near the inner angle of the cochlea over the labium vestibulare but growth in thickness here soon ceases. Next it develops rapidly over the cells which later line the spiral sulcus and form the inner supporting cells of the spiral organ. Finally, in later stages (yet to be described), vt grows rapidly over the spiral organ. From a study of my preparations 7t was not possible to demonstrate distinct fibers imbedded in a matrix nor are there grounds for believing that hairs or cilia take part in its development. 18.5 cm. stage. The later stages in the development of the cochlea show the further growth of the membrana over the spiral organ, its attachment to the latter, and the metamorphosis of the high columnar cells of the inner cell group to form the lining of the spiral sulcus. We have seen in earlier stages that differ- entiation of the cochlear duct is much more advanced in the basal coil than in the apical. This difference is very marked in a fetus of 18.5 em. In figure 14 the microphotograph shows sections of three turns on each side. The scalae are both large in the basal turn but in the upper turns the scala tympani is still small. The coagulated endolymph more or less completely fills the scala vestibuli. It will be seen when compared with the 13 em. stage that the membrana has continued to grow rapidly over the spiral organ in the two upper turns but its growth has ceased and it has remained small in the basal turn. Three stages in the development of the spiral sulcus and organ DEVELOPMENT OF THE MEMBRANA TECTORIA 443 are seen. In the upper turn (fig. 14, 3 and fig. 9) the epithelial cells just external to the teeth of the labium vestibulare have become lower, free from the membrana and tend to form a sim- ple epithelium. The space left between the membrana and the shortening cells is the spiral sulcus. The cells remaining between the spiral sulcus and the pillars of Corti still form a very high pseudostratified epithelium. In the middle turn (fig. 14, 2) the cells lining the spiral sulcus are Fig. 9 Section of 3d (upper) spiral of cochlear duct of the 18.5 cm. stage; 7.h.c., inner hair cells; 0.h.c., outer hair cells; lab.vest., labium vestibulare; limb.sp., spiral limbus; m.bas., membrana basilaris; m.tect., membrana tectoria; m.vest., membrana vestibularis; n.coch., cochlear nerve; sc.tymp., scala tympani; ft.sp., spiral tunnel. Oc. 4, Obj. 4, t. 1. 190. of the low columnar type with one or two rows of nuclei while the cells internal to the pillars are but little higher than the pillars themselves. Finally, in the lower turn (7) the cells lining the spiral sulcus are of the cubical type, in a single layer, and the remaining columnar cells persist as the internal supporting cells of the spiral organ. It seems probable that large numbers of the cells of the greater epithelial thickening degenerate, liquefy and disappear; those remaining flatten out and form the simple epithelium of the spiral sulcus. THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO. 4 444 Cc. W. PRENTISS In the apical turn, by comparing with figure 4 it will be seen that the membrana is but little thicker over the labium vestib- ulare, is much thicker over the inner cell group and thickest over the spiral organ where, in the 13 em. stage, it was just beginning to develop. It extends far beyond the outer support- ing cells. In the second turn the membrana is not so thick over the spiral organ but still extends beyond the outer hair cells. In the basal turn the membrana is thickest over the spiral sulcus and extends as far as the outer hair cells. In the two upper turns the membrana seems to be firmly attached to the cells of the greater epithelial thickening and to those of the spiral organ except along its outer zone where it shows signs of having shrunken and pulled away. In the basal turn the outer half was free but this also showed the effects of shrinkage and ~ distortion. Hardesty has suggested that there is a displacement of the spiral organ when the spiral sulcus is developed, thus accounting for the position of the membrana over the spiral organ in the later stages of its development. There are a number of facts which make this hypothesis untenable: (1) Sections and dissections of the 13 to 15 cm. stages show that the membrana is developed over the cells of the spiral organ. (2) In the 18.5 em. stage the membrana projects further beyond the spiral organ in the apical turn where the differen- tiation of the spiral sulcus has only just begun, and least in the lower turn where the spiral sulcus is fully developed. (3) The distance of the pillar cells from the inner angle of the cochlea (the only definite points which may be taken for comparative — measurements) is about the same in the 13 cm. and 18.5 cm. stages. (4) The total width of the thickened portion of the membrana is about one-fourth greater in the 18.5 cm. stage than in the 13 em. showing that growth has taken place along its outer border. This growth must have been supplied by the cells of the spiral organ. (5) To show that displacement takes place Hardesty measured the floor of the spiral sulcus and com- pared with the width of the inner cell group. There are no definite points which may be taken for measuring the floor of DEVELOPMENT OF THE MEMBRANA TECTORIA 445 the spiral suleus, and in measuring the width of the inner cell group one is including cells which form part of the spiral organ. No accurate comparison can thus be made. The distance be- tween the inner angle of the cochlea and the pillar cells, two definite points, may be measured with considerable accuracy and shows no important change in the position of the spiral organ from the 13 em. to the 18.5 cm. stage, nor later in the new-born animal. (6) As the basal membrane does not shorten, the dis- placement theory must assume that dead passive structures like the pillars actively move inward over the surface of the basal membrane. | One argument which Hardesty uses to prove that inward dis- placement of the spiral organ has occurred is that the ‘fibers’ of the membrana when traced from’its upper and outer border curve outward, downward, and then inward as though they had been pulled inward by the migration of the spiral organ. This inward curvature of the fibers is only found in the upper turns of the cochlea where the membrana is of greatest width and thickness. It may easily be accounted for. In the stages up to 16 em. the cells of the spiral organ slant outward but as the width of the basal membrane is rapidly increasing the inclina- tion of the chambers, hence of the ‘fibers’ is downward and outward. When the membrane begins to develop actively over the spiral organ the basal epithelium has attained its maximum width but as the cells are directed outward the inclination of the chambers will now be inward. When the spiral sulcus is developed by the degeneration of its cells, the outer cells of the spiral organ elongate and straighten somewhat so that they are no longer directed outward. This shifting, which is relatively slight and not. enough to account for the displacement of the membrana, would nevertheless increase the inward trend of its chambers. My observations are supported by those of Held (’09) on the guinea-pig and rabbit. In taking measurements of the 18.5 cm. stage the marked changes found are: (1) The increase in thickness of the membrana tectoria; (2) The increased distance from the inner angle of the cochlea to the labial teeth. The outer cells of the labium have 446 C. W. PRENTISS grown rapidly outward beneath the membrana thus pushing the ends of its chambers outward. The result is that in this region the chambers come to lie parallel to the surface of the labium and give the membrana a lamellated appearance which is espe- cially marked in the lower turns of the cochlea. The membrana may be divided into zones at this stage: (1) A thin structureless zone over the inner portion of the labium vestibulare; (2) A thicker second zone of flattened horizontal chambers over the outer portion of the labium vestibulare; (3) A still thicker third zone of chambers curving downward and outward unattached over the spiral suleus; (4) An outer zone, thickest in the upper turns with chambers trending downward, outward then inward, largely attached to the cells of the spiral organ and probably normally wholly thus attached. The sections of the 18.5 em. stage thus show that the mem- brana tectoria has developed rapidly over the spiral organ espe- cially in the upper turns of the cochlea; that the membrana is attached to the cells of the spiral organ in the upper coils and shows shrinkage and distortion in the lower; that the inner cells of the greater epithelial thickening degenerate or persist as the lining of the spiral sulcus while the outer cells of this group form the inner supporting cells of the spiral organ. Finally there is no evidence of an inward shifting of the spiral organ sufficient to account for the position of the membrana at this stage assum- ing (which we do not) that it is not developed from the spiral organ and that there is a necessity for such a displacement. The development of the structures arising from the basal epi- thelium of the cochlea is practically complete at 18.5 em., but a number of cochleae were studied from the full-term fetus. The structure of the membrana at this stage has been figured by Shambaugh (’07) and Hardesty (08) both of whom found attach- ments between the cells of the spiral organ and the membrana. These attachments are regarded as normal by many investi- gators, as due to coagulation and shrinkage by others. There is shown in figure 10 one of the many cases which occurred in my preparations showing attachment to the outer supporting cells. a DEVELOPMENT OF THE MEMBRANA TECTORIA 447 The membrana is undeniably shrunken and partly pulled away from the spiral organ, but the hairs of the outer auditory cells are firmly imbedded in the membrana. The under surface of the membrana shows a thickening, st.H., which according to Shambaugh (’08) corresponds to Hensen’s stripe and represents the inner line of its attachment to the inner supporting cells. In other sections the membrana was firmly attached to the inner supporting cells as well as to the hairs. In all of my prepara- tions at full-term the membrana was badly shrunken. Frequently \ lab. vest. WO) Mi) Fig.10 Section through the apical turn of the cochlea at about full term, show- ing outer auditory hairs imbedded in the membrana tectoria; ep.s.sp., epithelium of spiral sulcus; 7.h.c., inner hair cells; ¢.pil., inner pillar; m.bas., basal membrane; m.tect., membrana tectoria; lab.vest., labium vestibulare; limb.sp., limbus spir- alis; n.coch., cochlear nerve; 0.h.c., outer hair cell; sc.tymp., scala tympani; s.sp., Sulcus spiralis. Oc. 4, Obj. 4, t. 1. 190. also the organ of Corti was distorted, being pulled inward by the attached membrana. The attachment of the membrana to the spiral organ I regard as normal for the following reasons: 1. In development the membrana is normally so attached. 2. The auditory hairs when attached could be traced into the membrana, even though the latter was badly shrunken. 3. The shrinking membrane frequently exerts such a pull upon the spiral organ as to distort it. 448 Cc. W. PRENTISS 4. Physiologically and anatomically it is the condition we should logically expect if the membrana is functional in trans- mitting sound waves to the auditory hairs. 5. Were the membrana merely floating in contact with the hairs and unattached to them or their supporting cells it would not retain its position constantly and would thus interfere with the auditory function. In describing the structures of the cochlea the apex is regarded as above, the base as below and the membrana tectoria as lying ee Fig. 11 Dissection of the head of a pig fetus to show the position of the brain and cochlea. X 4. over the spiral organ. As a matter of fact when in its normal position the apex of the cochlea is directed cephalad and ven- trad. This may be well seen in a dissection of the brain and cochlea of the pig (fig. 11). When the pig’s snout is directed downward, as in feeding, the base of the cochlea would be above, the apex below. The same would be true of the human cochlea when the head is bent for- ward. The membrana tectoria would then be beneath the spiral organ and as it is shghtly heavier than the endolymph and very a DEVELOPMENT OF THE MEMBRANA TECTORIA 449 flexible it would naturally sink downward and away from the spiral organ assuming that it was not attached to the cells of the latter. This would be all the more apt to occur when the membrana is subjected to the heavy jars incident to active movements, running and jumping, and should interfere with hearing. As we know, such interference does not occur. The arguments raised by Hardesty against any normal attach- ment of the membrana, save to the labium vestibulare, are: 1. In dissecting the fresh membranous labyrinth to expose the membrana its outer portion could be seen floating free along its entire extent. 2. In the majority of sections it is entirely free from the spiral organ and when attached such attachments are filamentous and may be explained as abrasions of the under surface or coagula- tions of precipitated albumins. 3. “From the process of its development it seems probable that the membrane is free from the underlying structures,’ and as its outer zone acquires its position over the spiral organ by displacement, one must assume that any attach- ment which exists between the membrana and the spiral organ must have developed secondarily. Hardesty’s first argument bears little weight because in de- scribing his method of studying the fresh membrana tectoria he states that it was necessary to crush the bony labyrinth with a hammer and that the disturbances caused by his dissection caused the membrana to float free from the labium vestibulare an attach- ment which is never entirely ruptured in carefully fixed sections. A method which would destroy the strong attachment to the labium would certainly set free the more delicate attachments to the spiral organ. Moreover, dissections which were made by using more favorable methods did not seem to support Hardesty’s observations. As to the attachments seen in sections being artifacts it is sufficient to say that I have traced the hairs into the membrana in many eases and that attachments to the inner supporting cells and to the outer hair cells are so strong as to distort the spiral organ during the shrinkage of the membrana. The very fact that the ) 450 C. W. PRENTISS membrana shrinks shows that its normal position has been dis- turbed. We may as logically assume that it was attached and in many cases has shrunken away as to assume that it floated just parallel to the surface of the spiral organ and has become pressed down upon and attached to it by coagulations. We may assume this even more logically for we hold, and our preparations and dissections and the observations of Held (09) prove absolutely that the membrana is attached to the epithelial structures of the spiral organ in late fetal stages. There is no nec- essity for, and my preparations afford no proof of, an inward shifting of the spiral organ and a consequent displacement of the membrana. It is therefore unnecessary to assume with Hardesty and Von Ebner (’02) that any attachment between membrana and spiral organ must be of secondary development. While these arguments against the existence of an attachment between the membrana and spiral organ may be readily answered it is none the less true that a complete attachment to the cells of the spiral organ, such as exists in the fetus, has never been demonstrated in the adult organ. Nor, to my knowledge, has it been explained why the membrana should detach itself so readily from the spiral organ yet always retain its attachment to the labium vestibulare. First, as to the reason the complete attachment may be demonstrated in the early fetus and not in the adult: This is probably because the attachment is more firm in the fetus and because the basal epithelium and the basal membrane are less rigid in the fetus and tend to shrink pari passu with the membrana. In the adult or even the new-born young the tissues are less watery, more rigid and more resistant to reagents. The basilar membrane is attached to the bony laby- rinth, now strongly ossified. The membrana alone shrinks to any great extent and as a result is more or less completely torn away. Why the membrana should always lose its connection with the spiral organ and not its attachment to the labium vestibulare is explained by its structure. Over the labium it is an almost solid cuticular structure and the few chambers in this region are flat- tened and contain little fluid. Over the spiral sulcus the mem- DEVELOPMENT OF THE MEMBRANA TECTORIA 451 brana is composed of chambers, filled with fluid and open at their lower ends, while over the spiral organ these ends are assumed to be closed by the ends of the epithelial cells. The action of most fixing reagents and alcohol is to take water from the membrana. This would cause the open chambers to shrink, narrow, and so suddenly diminish the width and length of the membrana. As the membrana has the form of a spiral the shrinkage of the outer portion of the membrana throughout its whole length would tend to draw it toward the labium and away from the spiral organ, as it would diminish the diameter of the spiral. The effect would be most marked in the larger basal turns and it is there that the mem- brana is almost invariably torn away from the spiral organ even in late fetal stages. This alone would account for the detach- ment of the membrana from the spiral organ in most fixed prepa- rations. Over the spiral organ, assuming that the membrana is attached, the chambers would be closed by the ends of the epithe- lial cells. Upon the action of fixing reagents or alcohol, the with- drawal of water must take place chiefly about the ends of the chambers as their cuticular walls are not permeable. The result would be the shrinkage of the chambers and their separation from the cells. Even after the membrana is freed from its attachments Hardesty has shown that it shrinks very badly during the process of dehydration and clearing, and I have noted the same. The shrinkage of the membrana may also be aided by.normal tension in pulling the membrane away from the spiral organ. It is very possible that such tension exists especially in the lower turns of the cochlea, as held by Kishi (’07). THE FUNCTION OF THE MEMBRANA TECTORIA It is not my intention here to go into a detailed account of the physiology of audition but simply to emphasize certain anatomi- cal facts which have a bearing upon the transmission of the sound waves. Recent investigators all agree that the hair cells form the perceptive end organ of the cochlea and that the tectorial mem- brane is probably the medium through which the sound waves are transmitted to the hairs of the auditory cells. Arguments against 452 Cc. W. PRENTISS the old theory which regarded the basilar membrane as a reso- nator are many: 1. The structure of the basilar membrane, clothed as it is by several layers of cells, precludes its responding to delicate stimuli (Von Ebner ’02). 2: In the basal coil it is thick and rigid or may be replaced by a plate of bone though in this region the spiral organ is normally developed (Shambaugh ’07). 3. Hardesty has shown that the basilar membrane is merely a flattened tendon, the fiber bundles of which are closely bound together and thus could not vibrate separately. 4. The pillars are also rigidly united, and it is probable that the functions of the basilar membrane and of the pillars in con- junction with the lamina reticularis is to give rigidity to the auditory cells in order that their hairs may respond more readily to sound vibrations. . 5. The inner pillars do not always rest upon the basilar mem- brane but upon the edge of the labium tympanicum (Sham- baugh, Hardesty). 6. Sound waves entering the perilymph would affect the basilar membrane more strongly from the side of the scala tympani yet to do this would have to pass up and down the entire length of the spiral. The amplitude of the vibrations would be lessened by this and there’ would be interference between the waves going up in the scala vestibuli and the waves descending in the scala tympani. The objections raised against the basilar membrane do not apply to membrana, and there are many points in its favor: 1. The membrana is an exceeding delicate, chambered cuti- cular membrane, flexible yet elastic and of a specific gravity only slightly greater than that of the endolymph. 2. It is co-extensive with the spiral organ while the basilar membrane is not. 3. It lies on that side of the spiral organ at which sound waves would first enter the cochlea by way of the scala vestibuli. 4. It is attached along its inner edge to the labium vestibulare, stretches over the spiral sulcus and overlies the spiral organ in contact with and probably attached to its cells. DEVELOPMENT OF THE MEMBRANA TECTORIA 453 5. It is narrow and thin in the basal coil and becomes wider and thicker as it approaches the apex. Measurements of the func- tional zone of the membrana taken from its outer border to the labial teeth, show that the sectional area of the membrana in the apical turn is from thirty to forty times that in the basal turn. Owing to the shrinkage of the membrane such measurements ean be only approximate. Figure 12 shows the relative size of the membrana as seen in sections of the first, second, third and fourth turns. a a sa “Sate Fig. 12 Sections through the membrana tectoria of a full term fetus showing its relative size in the four turns. From Hardesty’s measurements of fresh and fixed preparations he found that the greatest width (first or apical turn) was about five times that of the minimal width (tip of basal turn) while the thickness in these same turns was as 6 : 1. The membrana tectoria is thus to be regarded as a spiral cuticular band of delicate chambered structure, which becomes eradually thicker and wider from base to apex. This band is attached by its flattened inner edge to the labium vestibulare, spans the spiral sulcus and its outer portion is so attached between the cells of the spiral organ that the ends of the cells close the chambers and the auditory hairs project into them. It may be 454 Cc. W. PRENTISS that the membrana is stretched with some tension between the labium and the spiral organ, the tension being greater at the base of the spiral. Whether a membrane of such structure and attach- ments may act as a whole or as a resonator, must be left to physicists to decide. There seems to be little doubt, however, that the membrana tectoria is the structure through which sound waves are transmitted to the auditory cells, and that it is in every way better adapted to this function than the basilar membrane. As it is thin, narrow and perhaps under tension in the basal turns and it has been shown that notes of high pitch are perceived here, it is probable that the membrana of the basal turns responds only to the sound waves of greatest frequency. While the apical turn, which receives notes of low pitch would respond only to waves of low frequency. It does not seem possible that any cuticular chamber could alone respond sympathetically to a given note but rather that a portion of the membrana, of nearly the same breadth and thickness, vibrates as a whole. SUMMARY 1. The membrana arises as a thin cuticular plate which is first developed over the free ends of the columnar cells which form the greater (inner) epithelial thickening of the basal cochlear wall. 2. As it is present in fetuses of 5 em., before the development of the hair cells in the spiral organ, it cannot be regarded as developed from these hairs. 3. The greater epithelial thickening gives rise to the epithelium of the labium vestibulare, to the lining of the spiral suleus and to the inner half of the spiral organ (inner supporting cells, and probably to the inner hair cells and inner pillars). The lesser epi- thelial thickening forms the external portion of the spiral organ. 4. The membrana grows in thickness by the secretion of a cuticulum formed between the ends of the epithelial cells, rapidly at first over the cells of the greater epithelial thickening (5 to 13 cm. stages), later over the cells of the lesser epithelial thick- ening. DEVELOPMENT OF THE MEMBRANA TECTORIA 455 5. In sections through the axis of the cochlea the membrana has a striated or lamellated appearance. The striae curve out- ward and downward from the labium vestibulare where the membrane remains thin. In sections perpendicular to the lamel- _lae the structures of the membrana is that of a reticulum with thickenings at the angles of the meshes. It is therefore neither lamellar nor reticular but a chambered structure or ‘honeycomb’ of hollow tapering cuticular tubes or chambers normally filled with a fluid resembling the endolymph. The bases of chambers: during development rest between the ends of the epithelial cells. 6. The thickenings at the angles of the meshes of the reticulum extend lengthwise along the whole extent of the tubes or chambers and in sections through the axis give the membrane its striated appearance, the striae having been variously interpreted as hairs, cilia, fibers and lamellae. 7. As the basal epithelium increases its width its cells are car- ried outward, away from the modiolus. This carries the bases of the growing cuticular chambers outward also, though their tips remain stationary. The result is the inward inclination of the chambers as they are followed from base to tip. 8. The chambered structure of the membrana explains the ‘border-plexus’ of Lowenberg, the accessory tectorial membrane observed by Hardesty, and the ‘reticular structure’ of the mem- brana described by various investigators. 9. In fetuses of 18.5 em., the membrana in the upper turns of the cochlea projects outward beyond the spiral organ and is firmly attached to the cells of both the spiral organ and of the greater epithelial thickening. In this turn the spiral sulcus has not yet fully formed and the distance from the inner angle of the cochlea to the pillars is fully as great as in the preceding stage. Thus the position of the membrana cannot be ascribed to an inward shifting of the spiral organ, but is due to its rapid develop- ment from the cells of the spiral organ. 10. The attachment of the membrana to the spiral organ was proved not only by sections but by dissections of both fresh and fixed cochleae. 456 Cc. W. PRENTISS 11. Between stages of 15 and 25 em., the inner cells of the greater epithelial thickening change from a high pseudostratified columnar type to that of a simple cubical epithelium. These cells lose their attachments to the membrana and the space which as high columnar cells they occupied, becomes the spiral sulcus. The change is brought about by the degeneration of many of the cells and the transformation of those remaining. 12. In sections of the cochlea at full term the membrana was found attached to the inner supporting cells of the spiral organ and to the outer hair cells as well as to the labium vestibulare. This attachment is regarded as normal because it was indicated by dissection of fresh cochleae; because in development it is so attached; because the attached membrane when shrinking under the action of reagents exerts such a pull upon the spiral organ as to distort it. Lastly, because physiologically and anatomically it is the condition which we should expect to find if the membrana is functional in transmitting sound waves to the auditory hairs. 13. Although usually described as lying above, the normal posi- tion of the membrana may be directly beneath the spiral organ. As it is slightly heavier than the endolymph if unattached it would float free especially when actively moved or jarred. This would interfere seriously with the function of the organ. 14. Reasons why the membrana detaches itself from the spiral - organ more readily than from the labium vestibulare are as follows: (a) The outer portion of the membrana being chambered shrinks much more than the inner zone which is a solid cuticular plate; (b) Shrinkage of the outer zone affects not only the width but the length of the membrana; (c) Being a spiral structure, the decrease in length decreases the diameter of the turns thus draw- ing the membrana inward. This would tend to separate it from its outer attachment to the cells of the spiral organ. 15. The arguments against regarding the basilar membrane as a medium for transmitting sound waves to the hair cells, do— not hold for the membrana tectoria. 16. The membrana tectoria is a delicate chambered cuticular structure, co-extensive with the spiral organ. It is attached by its inner zone to the labium vestibulare by its outer zone between DEVELOPMENT OF THE MEMBRANA TECTORIA 457 the cells of the spiral organ thus bridging over the spiral sulcus. Its sectional area at base and at apex is as 1 : 40 approximately. As the hairs of the auditory cells project directly into the chambers of the membrana, vibrations of the membrana would be directly transmitted to them. 17. As the membrana is much thinner and narrower in the basal turns than in the apical region it is probable that different por- tions of it respond to sounds of different pitch. In this sense it may act as a resonator. BIBLIOGRAPHY Ayers, H. 1891 Die membrana tectoria—was sie ist, und die membrana basil- aris—was sie verrichtet. Anat. Anz., Bd. 6. 1898 On the membrana basilaris, the membrana tectoria and the nerve endings in the human ear. Zool. Bull., vol. 1, no. 6. BorrcHer, A. 1870 Ueber Entwicklungsgeschichte und Bau des Gehérlaby- rinthes. Verhandl. der Kaiserl. Leop. Karol-deutschen Akad. der Natur- forseher, Bd. 35. Corti, A. 1851 Reserches sur l’organe de l’ouie des mammiferes. Zeitschr f. wiss Zoologie, Bd. 3. Coyne, P., pT Canntevu, A. 1895 Contribution 4 l’étude de la membran de Corti. Jour. de’l’anatomie et de la physiol., Ann. 31. Cz1NNER, H. J. unp Hammerscuuaa, V. 1898 Beitrag zur Entwicklungsge- schichte der Corti’schen membran. Archiv f. Ohrenheilk., Bd. 44. Von Exsner, V. 1902 In Kolliker’s Handbuch der Gewebelehre des Menschen, Bd. 3, 2d Halite. GOrrTsTEIN, J. 1872 Ueber den feineren Bau und die Entwicklung der Gehér- schnecke der Menschen und der Saiiger. Archiv f. mikr. Anat., Bd. 8. Harpesty, I. 1908 The nature of the tectorial membrane and its probable role in the anatomy of hearing. Amer. Jour. Anat., vol. 8. Hexup, H. 1909 Der feinerer Bau der Ohrlabyrinthes der Wirbelthiere. II. Abhandl. d. Sachs. Ges. d. Wiss. Math.-Phys. Kl. Bd. 31, pp. 191-293, Sian ate Von Hetmuoutz, H. L.T. 1896 Die Lehre von den Tonempfindungen. Ausgabe 5, Braunschweig. ; Kisar, K. 1907 Cortische Membran und Tonempfindungstheorie. Pfliiger’s Archiv, Bd. 116. K6uirker, A. 1861 Entwicklungsgeschichte des Menschen und der hdéheren Tiere, Leipsig. 458 Cc. W. PRENTISS LOWENBERG, B. 1864 Beitrige zur Anatomie der Schnecke. Archiv f. Ohren- heilk., Bd. 1. Retzius, G. 1884 Das Gehororgan der Wirbeltiere. Biologische Untersuchun- gen., Bd. 1, Stockholm. RicKENBACHER, O. 1901 Untersuchungen iiber die embryonale Membrana tec- toria der Meerschweinchen. Anat. Hefte, Abth. 1, Bd. 16. ScHWALBE, G. 1887 Lehrbuch der Anatomie der Sinnesorgane. SHAMBAUGH, G. E. 1907 A restudy of the minute anatomy of the structures in the cochlea, etc. Amer. Jour. Anat., vol. 7, no. 2. PLATE 1 EXPLANATION OF FIGURES Fig. 13 Microphotograph of an axial section through the cochlea of a 7.5 em. fetus. The numerals 1, 2, 3, indicate the turns of the spiral corresponding with those similarly numbered in figure 14. X 20. Fig. 14 Microphotograph of a section through the modiolus of a cochlea from an18.5em. fetus. Turns of cochlea numbered on right as in figure 13. XX 20. DEVELOPMENT OF THE MEMBRANA TECTORIA PLATE 1 Cc. W. PRENTISS THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO. 4 CHROMOSOMES IN MAN H. L. WIEMAN Zoological Laboratory, University of Cincinnati TEN FIGURES INTRODUCTION The literature dealing with the subject presents a wide range of possibilities in reaching conclusions regarding the nature and number of the chromosomes of man. Some of the earlier results may be dismissed, perhaps, on the ground of imperfect technic or unfavorable material, but the same means of elimination can not be used in removing difficulties in more recent observations. Bardeleben in 1892, the first to make any definite statement of the number of chromosomes in man, claimed it to be sixteen. The number twenty-four was first recorded by Flemming in 1897, if one may disregard the earlier work of Hansemann who also | found twenty-four in some cases, but in others eighteen and forty. Wilcox (’00) reported eighteen, with variations of fifteen and nineteen; but whether this represents the diploid or haploid number, he does not say. In 1906, Duesberg, on the basis of finding twelve chromosomes in the first spermatocyte division, corroborated Flemming’s conclusion that twenty-four is the unre- duced number. In the same year Moore and Arnold described sixteen gemini in the first spermatocyte metaphases, which would make the diploid number thirty-two. ! In 1910, Guyer published the observation of twenty-two chro- mosomes in the human spermatogonia. According to him, in _1In a study of the cytology of malignant growths in man, Farmer, Moore and Walker (Proc. Roy. Soc., B vol. 77, 1906) note the frequent occurrence in mitoses of 32 chromosomes which they consider the normal somatic comple- ment. 461 462 H. L. WIEMAN the growth period of the spermatocyte the nucleus contains two chromatin nucleoli of unequal size, and occasionally other small nucleolus-like granules which have no constancy in their pres- ence, size or relationship. When the spindle of the first sperma- tocyte forms, the chromatin nucleoli appear as accessory chro- mosomes, together with ten. bivalent chromosomes. The two accessories pass undivided to one pole of the spindle considerably in advance of the other chromosomes, with the result that one- half of the daughter cells in this division receive twelve, and the other half, ten univalent chromosomes. Since all the chromo- somes divide in the second spermatocyte division, one-half of the total number of spermatids receive twelve, and the other half, ten univalent chromosomes. Gutherz (12) working over the same ground, disagrees with Guyer’s interpretation of the two chromatin nucleoli appearing in the resting nucleus of the spermatocyte. Gutherz finds at this stage in addition to several (1 to 3) true nucleoli, a basophil nuc- leolus composed of a pair of chromosomes which at times assumes a quadripartite form. This tetrad-like appearance he takes to indicate a subsequent separation in both maturation divisions, which means an equal (quantitative) distribution of chromosomes to all spermatids. Gutherz does not describe the meiotic divi- sions, nor does he commit himself on the chromosome number. In his figure 10, of a first spermatocyte metaphase, twelve bodies may be counted, but it is by no means certain that they all repre- sent single chromosomes. He seems inclined to accept the results of Duesberg and of Branca (whose paper is not available to me at the present time), both of whom give twenty-four as the unreduced number. The unanimity of all recent work in settling on twenty-four, or a number very close to it (Guyer, 22 in the male), as the dip- loid number, is seriously disturbed by the results of Winiwarther (12) who reports the finding of forty-seven chromosomes in the spermatogonia. According to him, the metaphase of the first spermatocyte shows twenty-four chromosomes, one of which is a heterochromosome that later passes undivided to one pole of CHROMOSOMES iN MAN 463 the spindle during the division of the remaining twenty-three. In the second spermatocyte division figures, twenty-three and twenty-four chromosomes are seen, all of which divide; so that one-half of the spermatids receive twenty-three, and the other half, twenty-four. The difficulties of the problem of the chromosomes in man have not been lessened in any degree by the methods of attack. Thus the conclusions noted above were for the most part based on find- ings in the germ cells of the testis; while the other aspects of the problem, namely, that of the chromosomes in somatic mitoses, has been entirely neglected. It would appear that attempts at corroborating such conclusions by examination of somatic cells have proved unsatisfactory, and in most cases the somatic number has been arrived at by determining the number in the spermatogonia, or by multiplying the number observed in the maturation spindles by two. For the purpose of testing the reliability of this method as a criterion for determining the number of chromosomes in the soma- tic cells, as well as learning by direct observation something about the mitoses in these cells, I undertook the present study. This was made possible by the acquisition of unusually favorable material in the form of an apparently normal human embryo (white) measuring 9 mm. from crown to rump, which was fixed shortly after expulsion in an acetic-bichromate mixture, and cut in paraffinin to sections of 10. thickness.’ The sections were stained on the slide with Delafield hematoxylin and orange G, according to the method described by Morris (09). This stain- ing procedure proved an excellent one not only for general embryo- _ logical study, but for cytological as well, the chromosomes in many cases standing out with the sharpness of iron-alum-hema- toxylin preparations. ?For this embryo I amindebted to Dr. H. L. Woodward of Cincinnati, who obtained it from a case of abortion. 464 H., L. WIEMAN OBSERVATIONS Mitoses are abundant in every tissue of the body except the endoderm of the alimentary canal, the epidermis and the germ cells, most of which are in the resting condition. The germinative layer of the central nervous system shows the greatest number of division figures. The sex of course could not be determined. Prophases were found most favorable for study, and at this stage counts could be made with relative ease and accuracy, although in all cases the chromosomes lie at different levels. A scattering of the chromosomes throughout the cell is characteristic and sometimes a partial division with incomplete separation of the halves occurs before the metaphase. In selecting cells for illustration, I have considered only cells that are uncut by the knife and entirely included in the section. However, conclusions are based on the study of a much larger number, many of which might have been drawn. In many instances it is impossible to determine the number exactly owing to overlapping and crowding; but these are nevertheless useful when interpreted according to other cells indisputably clear. The number of the latter is not great, in spite of the large number of dividing cells in the material; for but relatively few combine the advantages of position in the section and sharp demarcation of the chromosomes from one another. Figure 1 is from a liver cell. The chromosomes, thirty-four in number, are in the form of rods, usually straight, but sometimes curved, or bent into V’s. No attempt has been made to pick out synaptic mates, but it can be readily seen that some of them fall into series of pairs, according to size and form (J, 2, 3). Figure 2 represents a remarkably clear prophase in a cell from the germinative layer of the brain in which the number of chromo- somes is thirty-three. The chromosomes are somewhat thicker than in the preceding cell. Figure 5 is also taken from the brain. This cell is ruptured so that the chromosomes are spread out as in a smear preparation, making the task of counting them a very. simple matter. The number is thirty-three or thirty-four, depend- ing upon whether or not we exclude the body marked z, which is —— Eee . CHROMOSOMES IN MAN 465 distinguished by its peculiar form and different level, from the other chromosomes. It is perhaps unnecessary to remark that all due precautions were taken in this case as well as others to avoid confusing the chromosomes lying in adjacent cells but dif- ferent sections. Figure 3 is from a mesenchyme cell lying directly beneath the epidermis of the ventral body wall, in which the form of the chromosomes differs somewhat from the preceding. At a and b are two thick heavy bars, and at e and d bodies resembling the bi- valent chromosomes of thé maturation spindles. The number is thirty-four, plus a small body z which may possibly be a plas- mosome fragment. Figure 4, a prophase of a cell in the mesothelium of the intes- tine, clearly shows thirty-four chromosomes, two to which are characterized by their small size and their location at opposite poles of the group (1). Figure 6 is a prophase figure from the mesenchyme of the lateral body wall in which the number of chromosomes is thirty-four. Figure 7, taken from a cell in the epithelium of the nasal pit, shows thirty-four chromosomes, many of them in the form of large thick rods. Figure 8 is from a neighboring cell in the nasal epithelium of the same section, in which the chromosomes are much smaller in size but larger in number, thirty-eight. It is possible that some of the chromosomes in this section are partially divided as the one at d, and that some of these halves were counted as single chromosomes. I have taken pains to avoid such an error, and I do not believe such a mistake was made; for in the case shown in figure 9, a mesenchyme eell from one of the visceral arches, in which the chromosomes are characterized by their small size, the number can be readily determined and is found to be thirty-eight. In connection with figure 3, attention was called to the bilobed appearance of several of the chromosomes. Della Valla, Gregoire and more recently Agar, have described similar chromosome- forms in somatic mitoses. When such a chromosome becomes split a tetrad-like body is formed as in figure 8,d. Hacker and later Schiller have brought about the formation of typical tetrads 466 H. L. WIEMAN The figures were outlined at table level by means of a camera lucida at the mag- nification obtained by using a 2 mm. apochromatic objective and compensating ocular 18 (Zeiss); while the details were drawn at a lower magnification. Figure 10 was later enlarged two diameters; all the figures were reduced } off in reproduction. CHROMOSOMES IN MAN 467 of 9 9.49, Fig. 1 Liver cell] 34 chromosomes. ‘ig. 2 Brain cell; 33 chromosomes. Fig. 3 Mesenchyme cell; 34 chromosomes. Fig. 4 Intestinal mesothelium cell; 34 chromosomes. Fig. 5 Brain cell (ruptured) ; 33, or 34 chromosomes if z is counted. Fig. 6 Mesenchyme cell; 34 chromosomes. Fig. 7 Nasal epithelium cell; 34 chromosomes. Fig. 8. Nasal epithelium cell; 38 chromosomes. Fig. 9 Mesenchyme cell; 38 chromosomes. Fig. 10 Enlarged drawing of chromosomes a,b,c,d and e, of figure 3. 468 H. L. WIEMAN in the diploid number by treating developing copepod eggs with ether; while Némec has caused their formation in plant tissue by means of chloral hydrate. Agar in his study of the chromosomes in larval Lepidosiren sums up his conclusions regarding this form of chromosome in these words: ‘‘The tendency for chromosomes to become transversely segmented or constricted is a wide-spread charac- teristic. It becomes especially operative, but not solely, whenever the chromosomes are short in comparison with their length as happens normally in meiosis and exceptionally in somatic tissue” (p. 295). One may readily find stages illustrating the steps in such a process. Figure 10 is an enlarged drawing of the chromosomes a,b,c,d and e of figure 3, which show how the segmentation might be brought about; a may be taken as the first step, the reduction in length resulting in a concentration of chromatic material at either end with a thinning out of the middle region. This thining out which gives the appearance of a transverse segmentation does not always occur in the middle, as may be seen from b. In ¢ the thin, or more lightly staining region is slightly constricted, and in s and e the constriction is well marked. The constriction does not represent a line of future division, for it can be clearly demon- strated that division takes place at right angles to it, that is, in a plane passing through the long axis of the chromosome. It is when such a segmented chromosome is beginning to divide that the tetrad-form is produced. DISCUSSION AND CONCLUSIONS In the foregoing I have described somatic mitoses in which thirty-three, thirty-four and thirty-eight chromosomes occur. . In addition to these I have observed other cases in which the num- ber is thirty-four. In still other instances it is impossible to state the number with certainty, but careful examination of several scores of mitoses leads me to believe that the number thirty-four approximates the one that occurs most frequently in the cells of the embryo under consideration. CHROMOSOMES IN MAN 469 The work of Duesberg, Guyer, Branea and Gutherz indicates that the pre-meiotic number is about twenty-four, and that the first spermatocyte metaphases contain one-half this number. My studies show clearly that in a human embryo the somatic mitoses display chromosomes in a number so much larger than this approxi- mate pre-metotic one, that the two numbers can not be the same. One may not always be able to determine with exactness the number of chromosomes, but when the number observed in a great many cases is ten more than the expected average, the values in the two cases must be different. It is a fact confirmed by countless observations that the number of chromosomes characteristic of the spermatogonia and the ovo- gonia, that is, the pre-meiotic number, is a constant one, and that the same is true of the meiotic division figures. On the other hand it is also known that the somatic mitoses de not always show a number identical with the pre-meiotic one. That this distine- tion is not generally recognized is evidenced by the frequency with which ‘somatic’ and ‘spermatogonial’ are used interchangeably, although in the classic Ascaris embryo the somatic cells undergo chromatin ‘diminution’ at the very beginning of cleavage. In Ascaris the chromosomes in the somatic cells are larger in number though smaller in size than in the germ cells. Krimmel (’10) finds the opposite condition in regard to number in the embryonic and somatic cells of the copepod, Diaptomus, in which the chromo- some number varies between the reduced and the diploid value. While it appears that the somatic number in man, though not a constant one perhaps, is different from the spermatogonial num- ber, one should not overlook the results of Moore and Arnold who observed sixteen bivalent chromosomes on the first spermatocyte spindle. The diploid number, thirty-two, would be one closely approaching the number found by me in the somatic cells. Their single figure of three first spermatocytes in division does not show sixteen bivalents in any case, nor does it support their claim in a very convincing manner. The results of Winiwarther are so at variance with all others that with the evidence at hand it is impossible to interpret them properly. It may be significant that the spermatogonial number 470 H. L. WIEMAN found by him, forty-seven, is about double that found by all recent observers, for this prompts the suggestion that in this case a doubling of chromosomes took place in early development. However, for the present this case must stand as an anomaly, and in view of the conclusions of all other workers in this field can not be accepted as representing a typical condition. The differences in the form and size of the chromosomes, as shown in the drawings, at first suggested a correlation between these characters and the tissues in which they were observed. Evidence for such an idea can be found in many cells, but is very much weakened by the fact that these distinguishing features are not constant, and the chromosomes of any tissue may appear differently under different conditions. Figures 8 and 9 show prophases containing thirty-eight chromo- somes, a number considerably above the average, thirty-four. Figures 7 and 8 are both from the epithelium of the nasal pit, the former showing thirty-four large chromosomes and the latter thirty-eight, many of which are much smaller. In figure 9 a number of the chromosomes are small in size. The same thing is true of a few other cases where I have observed a relatively large number of chromosomes in the somatic mitoses. These facts suggest that the small chromosomes may be derived by a break- ing up or ‘diminution’ of the larger ones. Likewise the differ- ence between the somatic number and the spermatogonial num- ber (as reported by Duesberg, Guyer and others) may have a similar explanation; but in view of the scanty and questionable character of the evidence, such an explanation can be offered only in a very tentative way. In order to throw more light upon this point, which is a highly important one in reaching any final con- clusions in assigning a proper value to the chromosomes in the organization of the cell, a comparative study of mitoses in embryos of the same and different ages, together with an examination of the maturation spindles, is necessary. I have made some head- way in securing material for this purpose, which will serve as the basis of a future study. CHROMOSOMES IN MAN 471 LITERATURE CITED Acar, W. E. 1912 Transverse segmentation and internal differentiation of chromosomes. Quart. Journ. Mic. Sc., No. 230, 58, Pt. 2. BARDELEBEN, K. von 1892 Ueber Spermatogenese bei Siugetieren. Verhandl. d. Anat. Gesellsch., Wien. Branca, A. 1912 Chractéres des deux mitoses de maturation chez homme. Cpts. rds. Assoc. des Anatom., 12 Réun. Bruxelles, (quoted from Gutherz). Deuita Vatua, P. 1908 Osservazioni di tetradi in cellule somatiche. Atti- della Reale Acad. d. Sci. Fis. e Mat. Napoli, 13. FLEMMING, VON W. 1897 Ueber die Chromosomenzahl beim Menschen. Anat. Anz., Bd. 15. GrecorrE, V. 1910 Les cinéses de maturation dans les deux régnes. (Second memoire) La Cellule, tom. 26. Guyer, M. F. 1910 Accessory chromosomes in man. Biol. Bull., vol. 19. GUTHERZ, S. 1912 Uber ein bemerkenswertes Strukturelement (Heterochromo- some?) in der Spermiogenese des Menschen. Arch. fiir Mic. Anat., Bd. 79. Hacker, V. 1900 Mitosen in Gefolge Amitosenihnlicher Vorginge. Anat. Anz., Bd. 17. 1912 Ergebnisse und Ausblicke in der Keimzellforschung. Zeitschr. fur Abstam. u. Vererbungslehre., Bd. 7. Krimer, O. 1910 Chromosomenverhiltnisse in generativen und somatischen Mitosen bei Diaptomus coeruleus nebst Bemerkungen iiber die Ent- wickelung der Geschlechtsorgane. Zool. Anz., Bd. 35. Moorsg, J. E. S., AnD ARNoLpD, G. 1906 On the existence of permanent forms among the chromosomes of the first meiotic division in certain animals. Proc. Roy. Soc., B 77. Morris, J. T. 1909 A note on orange G counter-staining suggesting a useful method in the management of embryonic tissue. Anat. Rec. vol. 3. Scuitier, I. 1909 Uber kunstliche Erzeugung ‘primitiver’ Kernteilungsformen bei Cyclops. Arch. fiir Entwicklungsmech., Bd. 27. Witcox, E. V. 1900 Human spermatogenesis. Anat. Anz., 17. WInrwarTHER, H. von 1912 Etudes sur la spermatogenése humaine. Arch. d. Biol., Bd. 27. THE SPERMIOGENESIS OF THE PRIBILOF FUR SEAL (CALLORHINUS ALASCANUS J. AND C.)! JEAN REDMAN OLIVER The Histological Laboratory of the Department of Physiology and Histology, Leland Stanford Junior University, California THIRTY-BIGHT FIGURES Notwithstanding the considerable number of investigators, who have studied the spermatogenesis of the mammalia in recent years, many questions remain undecided, and the number of species examined is still but small. The domestic animals and the more common wild forms have furnished practically all the material thus far, and any addition to this number would seem very desirable. The following account of the spermiogenesis in the fur seal, Callorhinus alascanus Jordan and Clark, is offered as such a contribution. The material, assigned to me by Prof. F. M. MacFarland for this study, was preserved in the Pribilof Islands, Alaska, by Mr. G. A. Clark, Special Assistant in Charge of Fur Seal Investi- gations during the summer of 1909, to whom I desire to extend my sincere thanks for his kind co-operation. Four different fixatives had been used, namely, saturated corrosive sublimate, Bouin’s picro-formalin-acetic mixture, Lenhossék’s sublimate- alcohol-acetic, and strong Flemming’s solution. Small pieces of the fresh testes of two fur seals, one a young male of three years, the other a fully grown adult of eight years of age, were fixed in each of these reagents. All stages in the development of the spermatozoa were found in material from each individual and no important differences were noted, so that this paper is based upon the study of preparations made from both. The sublimate material was dehydrated and iodinized in the usual way and, 1 Published by permission of the Hon. George N. Bowers, U. S. Commissioner of Fisheries, Washington, D. C. 473 474 JEAN REDMAN OLIVER alter paraffin imbedding, sections 5 « were cut. This was as thin as was found practical, and in most cases was found sufficient for all purposes. After mounting on the slide the sections were treated with a 24 per cent solution of sodium thiosulphate, diluted with ten volumes of water, to remove all traces of iodine, as recommended by Heidenhain (’09). As for stains, though many others were used, Heidenhain’s iron hematoxylin was found by far the most satisfactory, all the elements of the cells being shown in a most favorable manner. The ordinary treatment was used with a slightly longer period of mordantage, the sections remaining in the iron bath for twelve hours at least, and then from twelve to twenty-four hours in the stain. For contrast stains erythrosin, eosin, congo red and orange G were mainly used, the first named giving the best results as it was less likely to overstain and thus obscure delicate structures. This I found to be a serious danger as a heavy stain is likely to cause delicate fila- ments, such as are seen in the origin of the ‘Sehwanzmanschette’ or caudal tube, or the centrosomes even, to be overlooked. For the study of the mature spermatozoa cover glass smear prepa- rations were made from the epididymis, which has been pre- served as a whole in 3 per cent formaldehyde. Further prepa- rations were also made from new material secured by Mr. Clark in the summer of 1912, and the whole development was verified with these. THE DIVISIONS OF SPERMIOGENESIS In describing the process of the development of the adult sperm from the spermatid I shall follow the plan first laid down by Meves (’99) who separated the process into four main periods. The first extends from the close of the division of the sperma- tocytes of the [Td order to form the spermatids, up to the appear- ance of the ‘Schwanzmanschette,’ or caudal tube; the second extends from this appearance to its final disappearance; the third period from this point up to the migration of the adult sperm into the lumen of the tubule; and the fourth period, that of the so-called ‘maturation,’ consisting of minor changes in form and density mainly, which take place in the epididymis. SPERMIOGENESIS OF THE PRIBILOF FUR SEAL 475 The first period: from the second dimsion of the spermatocytes up to the appearance of the caudal tube: figures 1 to 15 In describing the changes which the spermatid undergoes, there are at least four principal structures to be noticed, the cytoplasm, the nucleus, the idiosome, and the centrosomes. Other important structures appear later and will be mentioned as they occur. Immediately after the second division of the spermatocytes, the small spermatids are found along the inner surface of the epithelial lining of the tubule (fig. 1). As a rule each is poly- hedral, due to the pressure of the adjacent cells. Those sper- matids lying nearest the lumen of the tubule appear less influ- enced by this factor and are often quite rounded. The cytoplasm is clear and transparent, or but slightly granular, and is bounded by a sharply defined cell membrane. The spherical nucleus is at first central in position, but as development proceeds, the whole spermatid becomes distally elongated toward the lumen of the tubule, and the nucleus shifts in position toward the proximal end of the cell. It is comparatively large, resembling both in size and form that of the Von Ebner cells which precede them. So marked is this resemblance that it is often difficult to distin- guish between these two stages. The chromatin is scattered, as a rule, throughout the whole area of the nucleus in the form of large irregular clumps which are connected by slender bands of colorless linin (figs. 1 to 5). The nuclear membrane is quite clear and distinct and is generally incrusted on its inner surface with a layer of chromatin of varying thickness. In the closing portion of this period the chromatin begins to change toward the appearance which it has in the adult sperm. The large irregular clumps become resolved into finer granules which are more evenly distributed over the linin network, the nucleus as a whole losing its mottled appearance and taking on a more homogeneous aspect (figs. 11 to 14). As the end of the period approaches the nucleus also elongates toward the wall of the tubule and shifts bodily in that direction, finally coming to lie at the extreme proximal end of the cell THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO. 4 476 JEAN REDMAN OLIVER body (figs. 13 to 15) toward the tubule wall. The prolongation, described by Duesberg (’08) in the spermatid of the rat, which the nucleus sends out to meet the centrosomes is not visible in the fur seal. In the second period the nucleus is often elongated in such a manner, but it is long after the centrosomes have reached the nuclear membrane. The idiosome, or sphere, of the fur seal shows in most respects those characteristics which have been described by previous authors in mammalian spermiogenesis. It appears shortly after the second division of the spermatocytes as a homogeneous body of considerable size, lying close to the nuclear wall, and usually near its proximal end. It takes an eosin stain a little more deeply than the surrounding cytoplasm, and shows no traces of the cortical granulations or other structural differentiations de- scribed for some forms. In shape it is not strictly a sphere but has more the form of a prolate ellipsoid (fig. 1, s). The testis of the fur seal is not as favorable material for the study of the development of the acrosome as that of some other forms, but the course of events can be readily followed, and differs but slightly from that described by Benda (’91), Moore (’94), Niessing (96), Lenhossék (’98), Meves (’99) and others. The first change in the homogeneous idiosome that I have been able to find is the relatively sudden appearance of two densely staining gran- ules, lying in a clearer area within it (fig. 2). The whole sphere stains very faintly at this stage and appears hyaline and semi- transparent. These two granules apparently fuse into a single dense body in the center of the hyaline area (fig. 3). Not all of the sphere is made up of this hyaline substance, however, a denser and more opaque portion lying at one side (fig. 4, s.r.) and gradually separating entirely from the clear portion, destined to form the ‘capuchon,’ or head cap of the mature spermatozoon. This darker remnant of the sphere migrates to the distal part of the cell (figs. 13 to 15) where it finally degenerates along with the cytoplasm of that region, and is cast off in the closing stages. The hyaline body is at first spherical (figs. 3 and 4) but soon begins to flatten against the nuclear wall, its contained central granule, the acrosome, coming in contact with the latter and SPERMIOGENESIS OF THE PRIBILOF FUR SEAL A477 likewise flattening against it. This fusion becomes so intimate that it is often impossible to distinguish the acrosome in the later stages of the spermatid. To what the striking variations in size of the acrosome, such as are shown in figures 5 to 9, may be due I am unable to decide. In the early stages of the contact of the hyaline body with the nucleus the wall of the latter frequently shows a marked degree of flattening, as though yielding to an external pressure, and in many cases the line of contact becomes actually concave (figs. 5 to 7). This condition is, however, but transitory and the convex outline is soon resumed. A marked change also occurs in the substance of the hyaline body now extending back over the nuclear wall. Its substance becomes more dense anteriorly, and stains more readily with eosin. This process continues back- ward toward the nuclear membrane until the whole head cap becomes transformed into this denser substance. Figure 9 shows a midway stage in which the more anterior part of the head cap has become dense, while the part next to the nuclear mem- brane is still hyaline. Figures 11 to 15 show the head cap en- tirely composed of the denser substance. During this differ- entiation the process of flattening and overgrowth is continuing, so that by the end of the first period the head cap has extended well down over the anterior half of the nucleus. It is manifest from the above that the head cap reaches nearly its adult form in the first period of spermiogenesis, and its subsequent develop- ment consists mainly in a process of further differentiation, no new elements being added to the form which it has already assumed. The first appearance of the centrosomes in the young spermatid is at the periphery of the cell, in the form of two small, rounded granules, one a little larger than the other, lying close to the cell membrane (fig. 1, c.). Actual proof of the centrosomal nature of these granules in the fur seal is lacking, as I have not traced their earlier history. From subsequent events, however, and from analogy with other forms in which their history is fully known, I deem it safe to consider them as the centrosomes. In the earliest stage in which I have been able to detect them there 478 JEAN REDMAN OLIVER is no trace of a tail filament. It soon appéars, however (fig. 10, a.f.) and may be readily found in cases in which the centro- somes have migrated inward to their contact with the nuclear membrane, as in figures 11 and 12. In nearly every instance the tail filament projects out freely beyond the boundary of the cell, and even in such an early stage as that shown in figure 10, it has already reached a considerable length. The appearance and growth of the tail filament must be extremely rapid, for it is very difficult to find any early stages. The migration of the centrosomes toward the nucleus must be quite rapid also, as intermediate stages between those shown in figures 10 and 11 are very rare. No indication of any prolongation extending from the nucleus toward the centrosomes, such as described by Meves (99), and by Duesberg (’08), could be found, and the migration seems to be entirely an active one on the part of the centrosomes, so far as such a visible participation of the nucleus is concerned. After reaching the nucleus the anterior centrosome becomes closely pressed up against the membrane (figs. 11 and 12) and at the end of the first period it is fused with the membrane, often almost disappearing from view in the chromatin incrusted wall. In this fusion the anterior centrosome is lengthened in a direction at right angles to the tail filament. In many instances it appears as if the centrosome had penetrated the membrane and was situated on its inner surface as in figures 13 and 14. The second period: from the appearance of the caudal tube to the migration of the distal half of the posterior centrosome (the annu- lus) along the tail filament: figures 16 to 29 , In the original division of Meves (’99) the second period extends from the origin of the ‘Manschette’ up to its total disappearance from the cell. As will be shown in the following pages the ‘Manschette’ does not disappear in the developing spermatid of the fur seal, but persists and takes an important place in the structure of the adult sperm. For this reason we are compelled to seek another phenomenon which, if possible, occurs constantly at the time of the disappearance of the caudal tube in other — SPERMIOGENESIS OF THE PRIBILOF FUR SEAL 479 forms, that we may limit the present period in our material. Meves (’99) in his work on-the guinea-pig states that the dis- appearance of the ‘Manschette’ is synchronous with the beginning of the movement of the annulus along the tail filament, a state- ment later confirmed for the rat by Duesberg (’08). The general parallelism of events in the fur seal spermiogenesis with that in other mammals leads us to consider this migration of the annulus down the tail filament as marking the close of the second period. Retzius (09, p. 227), has well pointed out that the term ‘Manschette’ is not a well selected name for a structure around a neck, and that ‘Halskragen’ is not much better, since both imply an opening at one side, either open or buttoned. As the structure in question is a true tube, he prefers the older name ‘Schwanzrohre,’ which I have adopted in this description in the translated form ‘caudal tube.’ The cytoplasmic body of the spermatid at the beginning of this period is still more or less polygonal in section, with the nucleus more or less shifted proximally from its central position. During this stage the whole cell becomes elongated in a direction radial to the tubule, and the nucleus approaches the proximal end of the cell until it reaches the surface, the cytoplasm being massed toward the lumen while the cell membrane and acrosome cover the apical portion of the nucleus. 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